Genetic sequences encoding flavonoid pathway enzymes and uses therefor

ABSTRACT

The present invention relates generally to genetic sequences encoding flavonoid pathway metabolising enzymes and more particularly to flavonoid 3′-hydroxylase (hereinafter referred to as “F3′H”) or derivatives thereof and their use in the manipulation of pigmentation in plants and other organisms.

The present invention relates generally to genetic sequences encodingflavonoid pathway metabolising enzymes and more particularly toflavonoid 3′-hydroxylase (hereinafter referred to as “F3“H”) orderivatives thereof and their use in the manipulation of pigmentation inplants and other organisms.

Bibliographic details of the publications referred to by the author inthis specification are collected at the end of the description. SequenceIdentity Numbers (SEQ ID NOs) for the nucleotide and amino acidsequences referred to in the specification and claims are definedfollowing the bibliography. A summary of the SEQ ID NOs, and thesequences to which they relate, is provided prior to the Examples.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The rapidly developing sophistication of recombinant DNA technology isgreatly facilitating research and development in a range ofbiotechnology related industries. The horticultural industry has becomea recent beneficiary of this technology which has contributed todevelopments in disease resistance in plants and flowers exhibitingdelayed senescence after cutting. Some attention has also been directedto manipulating flower colour.

The flower industry strives to develop new and different varieties offlowering plants. An effective way to create such novel varieties isthrough the manipulation of flower colour. Classical breeding techniqueshave been used with some success to produce a wide range of colours formost of the commercial varieties of flowers. This approach has beenlimited, however, by the constraints of a particular species' gene pooland for this reason it is rare for a single species to have a fullspectrum of coloured varieties. In addition, traditional breedingtechniques lack precision. The aesthetic appeal of the flower is acombination of many factors such as form, scent and colour; modificationof one character through hybridization can often be at the expense of anequally valuable feature. The ability to genetically engineer precisecolour changes in cutflower and ornamental species would offersignificant commercial opportunities in an industry which has rapidproduct turnover and where novelty is an important marketcharacteristic.

Flower colour is predominantly due to two types of pigment: flavonoidsand carotenoids. Flavonoids contribute to a range of colours from yellowto red to blue. Carotenoids impart an orange or yellow tinge and arecommonly the major pigment in yellow or orange flowers. The flavonoidmolecules which make the major contribution to flower colour are theanthocyanins which are glycosylated derivatives of cyanidin, delphiidin,petunidin, peonidin, malvidin and pelargonidin, and are localised in thevacuole. The different anthocyanins can produce marked differences incolour. Flower colour is also influenced by co-pigmentation withcolourless flavonoids, metal complexation, glycosylation, acylation andvacuolar pH (Forkmann, 1991).

The biosynthetic pathway for the flavonoid pigments (hereinafterreferred to as the “flavonoid pathway”) is well established and is shownin FIGS. 1 a and 1 b (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach,1979; Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990;Van Tunen and Mol, 1990; Dooner et al, 1991; Martin and Gerats, 1993;Holton and Cornish, 1995). The first committed step in the pathwayinvolves the condensation of three molecules of malonyl-CoA with onemolecule of p-coumaroyl-CoA. This reaction is catalysed by the enzymechalcone synthase (CHS). The product of this reaction, 2′,4,4′,6′,tetrahydroxy-chalcone, is normally rapidly isomerized to producenaringenin by the enzyme chalcone flavanone isomerase (CHI). Naringeninis subsequently hydroxylated at the 3 position of the central ring byflavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).

The pattern of hydroxylation of the B-ring of DHK plays a key role indetermining petal colour. The B-ring can be hydroxylated at either the3′, or both the 3′ and 5′ positions, to produce dihydroquercetin (DHQ)and dihydromyricetin (DHM), respectively. Two key enzymes involved inthis pathway are flavonoid 3′-hydroxylase and flavonoid3′,5′-hydroxylase, both of the cytochrome P450 class. Cytochrome P450enzymes are widespread in nature and genes have been isolated andsequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.

Flavonoid 3′-hydroxylase acts on DHK to produce DHQ and on naringenin toproduce eriodictyol. Reduction and glycosylation of DHQ produces thecyanidin-glycoside and peonidin-glycoside pigments which, in many plantspecies (for example rose, carnation and chrysanthemum), contribute tored and pink flower colour. The synthesis of these anthocyanins can alsoresult in other flower colours. For example, blue cornflowers containcyanin. The ability to control flavonoid 3′-hydroxylase activity, orother enzymes involved in the flavonoid pathway, in flowering-plantswould provide a means to manipulate petal colour. Different colouredversions of a single cultivar could thereby be generated and in someinstances a single species would be able to produce a broader spectrumof colours.

A nucleotide sequence (referred to herein as SEQ ID NO:26) encoding apetunia flavonoid 3′-hydroxylase has been cloned (see InternationalPatent Application No. PCT/AU93/00127 [WO 93/20206]). However, thissequence was inefficient in its ability to modulate the production of3′-hydroxylated anthocyanins in plants. There is a need, therefore, toidentify further genetic sequences encoding flavonoid 3′-hydroxylaseswhich efficiently modulate the hydroxylation of flavonoid compounds inplants. More particularly, there is a need to identify further geneticsequences encoding flavonoid 3′-hydroxylases which efficiently modulatethe production of 3′-hydroxylated anthocyanins in plants.

In accordance with the present invention, genetic sequences encodingflavonoid 3′-hydroxylase have been identified and cloned. Therecombinant genetic sequences of the present invention permit themodulation of expression of genes encoding this enzyme by, for example,de novo expression, over-expression, suppression, antisense inhibitionand ribozyme activity. The ability to control flavonoid 3′-hydroxylasesynthesis in plants permits modulation of the composition of individualanthocyaains as well as alteration of relative levels of flavonols andanthocyanins, thereby enabling the manipulation of tissue colour, suchas petals, leaves, seeds and fruit. The present invention is hereinafterdescribed in relation to the manipulation of flower colour but this isdone with the understanding that it extends to manipulation of otherplant tissues, such as leaves, seeds and fruit.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides encoding aflavonoid 3′-hydroxylase or a derivative thereof wherein said flavonoid3′-hydroxylase or its derivative is capable of more efficient modulationof hydroxylation of flavonoid compounds in plants than is a flavonoid3′-hydroxylase encoded by the nucleotide sequence set forth in SEQ IDNO:26.

Efficiency as used herein relates to the capability of the flavonoid3′-hydroxylase enzyme to hydroxylate flavonoid compounds in a plantcell. This provides the plant with additional substrates for otherenzymes of the flavonoid pathway able to further modify this molecule,via, for example, glycosylation, acylation and rhamnosylation, toproduce various anthocyanins which contribute to the production of arange of colours. The modulation of 3′-hydroxylated anthocyanins isthereby permitted. Efficiency is conveniently assessed by one or moreparameters selected from: extent of transcription, as determined by theamount of mRNA produced; extend of hydroxylation of naringenin and/orDHK; extent of translation of mRNA, as determined by the amount oftranslation product produced; extent of production of anthocyaninderivatives of DHQ or DHM; the extent of effect on tissue colour, suchas flowers, seeds, leaves or fruits.

Another aspect of the present invention is directed to an isolatednucleic acid molecule comprising a sequence of nucleotides which maps tothe genetic locus designated Ht1 or Ht2 in petunia, or to equivalentsuch loci in other flowering plant species, and wherein said isolatednucleic acid molecule encodes, or is complementary to a sequence whichencodes, a flavonoid 3′-hydroxylase.

A further aspect of the present invention contemplates an isolatednucleic acid molecule comprising a sequence of nucleotides whichcorresponds to the genetic locus designated Ht1 or Ht2 in petunia, or toloci in other flowering plant species which contain sequences whichcontrol production of 3′-hydroxylated flavonoids, and wherein saidisolated nucleic acid molecule encodes a flavonoid 3′-hydroxylase or aderivative thereof which is capable of more efficient conversion of DHKto DHQ in plants than is the flavonoid 3′-hydroxylase set forth in SEQID NO:26.

In accordance with the above aspects of the present invention there isprovided a nucleic acid molecule comprising a nucleotide sequence orcomplementary nucleotide sequence substantially as set forth in SEQ IDNO: 1 or having at least about 60% similarity thereto or capable ofhybridising to the sequence set forth in SEQ ID NO: 1 under lowstringency conditions.

In a related embodiment, there is provided a nucleic acid moleculecomprising a nucleotide sequence or complementary nucleotide sequencesubstantially as set forth in SEQ ID NO:3 or having at least about 60%similarity thereto or capable of hybridising to the sequence set forthin SEQ ID NO:3 under low stringency conditions.

In another related embodiment, the present invention is directed to anucleic acid molecule comprising a nucleotide sequence or complementarynucleotide sequence substantially as set forth in SEQ ID NO:5 or havingat least about 60% similarity thereto or capable of hybridising to thesequence set forth in SEQ ID NO:5 under low stringency conditions.

Yet another related embodiment provides a nucleic acid moleculecomprising a nucleotide sequence or complementary nucleotide sequencesubstantially as set forth in SEQ ID NO:7 or having at least about 60%similarity thereto or capable of hybridising to the sequence set forthin SEQ ID NO:7 under low stringency conditions.

Still yet a further embodiment of the present invention relates to anucleic acid molecule comprising a nucleotide sequence or complementarynucleotide sequence substantially as set forth in SEQ ID NO:9 or havingat least about 60% similarity to the coding region thereof or capable ofhybridising to the sequence set forth in SEQ ID NO:9 under lowstringency conditions.

In another further embodiment, there is provided a nucleic acid moleculecomprising a nucleotide sequence or complementary nucleotide sequencesubstantially as set forth in SEQ ID NO: 14 or having at least about 60%similarity thereto or capable of hybridising to the sequence set forthin SEQ ID NO: 14 under low stringency conditions.

In yet another further embodiment, the present invention is directed toa nucleic acid molecule comprising a nucleotide sequence orcomplementary nucleotide sequence substantially as set forth in SEQ IDNO: 16 or having at least about 60% similarity thereto or capable ofhybridising to the sequence set forth in SEQ ID NO: 16 under lowstringency conditions.

Still yet another further embodiment provides a nucleic acid moleculecomprising a nucleotide sequence or complementary nucleotide sequencesubstantially as set forth in SEQ ID NO:18 or having at least about 60%similarity thereto or capable of hybridising to the sequence set forthin SEQ ID NO: 18 under low stringency conditions.

Moreover, yet a further embodiment of the present invention relates to anucleic acid molecule comprising a nucleotide sequence or complementarynucleotide sequence substantially as set forth in SEQ ID NO:20 or havingat least about 60% similarity thereto or capable of hybridising to thesequence set forth in SEQ ID NO:20 under low stringency conditions.

Still yet another further embodiment is directed to a nucleic acidmolecule comprising a nucleotide sequence or complementary nucleotidesequence substantially as set forth in SEQ ID NO:22 or having at leastabout 60% similarity thereto or capable of hybridising to the sequenceset forth in SEQ ID NO:22 under low stringency conditions.

In still yet another further embodiment, the present invention providesa nucleic acid molecule comprising a nucleotide sequence orcomplementary nucleotide sequence substantially as set forth in SEQ IDNO:24 or having at least about 60% similarity thereto or capable ofhybridising to the sequence set forth in SEQ ID NO:24 under lowstringency conditions.

In a particularly preferred embodiment there is provided an isolatednucleic acid molecule comprising a nucleotide sequence or complementarynucleotide sequence substantially as set forth in SEQ ID NO: 1 or havingat least about 60% similarity thereto or capable of hybridising to thesequence set forth in SEQ ID NO: 1 under low stringency conditions,wherein said nucleotide sequence maps to the genetic locus designatedHt1 or Ht2 in petunia, or to equivalent such loci in other floweringplant species, and wherein said isolated nucleic acid molecule encodes,or is complementary to a sequence which encodes, a flavonoid3′-hydroxylase.

Reference herein to a low stringency at 42° C. includes and encompassesfrom at least about 1% to at least about 15% formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% to atleast about 30% formamide and from at least about 0.5 M to at leastabout 0.9 M salt for hybridization, and at least about 0.5 M to at leastabout 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% to at least about 50%formamide and from at least about 0.01 M to at least about 0.15 M saltfor hybridization, and at least about 0.01 M to at least about 0.15 Msalt for washing conditions. Hybridization may be carried out atdifferent temperatures and, where this occurs, other conditions may beadjusted accordingly.

Another aspect of the present invention provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO:2 or an amino acid sequence having at least about 50%similarity thereto.

In a related embodiment, there is provided a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO:4 or an amino acid sequence having at least about 50%similarity thereto.

A further related embodiment of the present invention is directed to anucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in SEQ ID NO:6 or an amino acid sequencehaving at least about 50% similarity thereto.

Still another related embodiment provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO:8 or an amino acid sequence having at least about 50%similarity thereto.

Yet still another related embodiment relates to a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or anamino acid sequence having at least about 50% similarity thereto.

In another further embodiment, there is provided a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO: 15 or an amino acid sequence having at least about 50%similarity thereto.

In yet another further embodiment, the present invention is directed toa nucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in SEQ ID NO: 17 or an amino acid sequencehaving at least about 50% similarity thereto.

Still yet another further embodiment provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inSEQ ID NO: 19 or an amino acid sequence having at least about 50%similarity thereto.

Moreover, yet a further embodiment of the present invention relates to anucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in SEQ ID NO:21 or an amino acid sequencehaving at least about 50% similarity thereto.

Still yet another further embodiment is directed to a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding an amino acid sequence substantially as set forthin SEQ ID NO:23 or an amino acid-sequence having at least about 50%similarity thereto.

In still yet another further embodiment, the present invention providesa nucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in SEQ ID NO:25 or an amino acid sequencehaving at least about 50% similarity thereto.

In a particularly preferred embodiment there is provided an isolatednucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in SEQ ID NO:2 or an amino acid sequencehaving at least about 50% similarity thereto, wherein said sequence ofnucleotides maps to the genetic locus designated Ht1 or Ht2 in petunia,or to equivalent such loci in other flowering plant species, and whereinsaid isolated nucleic acid molecule encodes, or is complementary to asequence which encodes, a flavonoid 3′-hydroxylase or a derivativethereof.

The term “similarity” as used herein includes exact identity betweencompared sequences, at the nucleotide or amino acid level. Where thereis non-identity at the nucleotide level, “similarity” includesdifferences between sequences which result in different amino acids thatare nevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels.

The nucleic acid molecule defined by SEQ ID NO: 1 encodes a flavonoid3′-hydroxylase from petunia. Examples of other suitable F3′H genes arefrom carnation (SEQ ID NO:3), snapdragon (SEQ ID NO:5), arabidopsis (SEQID NO:7), arabidopsis genomic DNA clone (SEQ ID NO: 9), rose (SEQ ID NO:14), chrysanthemum (SEQ ID NO:16), torenia (SEQ ID NO: 18), Japanesemorning glory (SEQ ID NO:20), gentian (SEQ ID NO:22) and lisianthus (SEQID NO:24). Although the present invention is particularly exemplified bythe aforementioned F3′H genes, the subject invention extends to F3′Hgenes from any species of plant provided that the F3′H gene has at leastabout 60% similarity at the nucleotide level to a nucleic acid moleculeselected from SEQ ID NO: 1 or 3 or 5 or 7 or 14 or 16 or 18 or or 22 or24, or at least about 50% similarity at the amino-acid level to an aminoacid molecule selected from SEQ ID NO: 2 or 4 or 6 or 8 or 10, 11, 12,13 or 15 or 17 or 19 or 21 or 23 or 25. The subject invention furtherextends to F3′H genes from any species of plant provided that the F3′Hgene has at least about 60% similarity at the nucleotide level to thecoding region of SEQ ID NO:9.

The nucleic acid molecules of the present invention are generallygenetic sequences in a non-naturally-occurring condition. Generally,this means isolated away from its natural state or synthesized orderived in a non-naturally-occurring environment. More specifically, itincludes nucleic acid molecules formed or maintained in vitro, includinggenomic DNA fragments, recombinant or synthetic molecules and nucleicacids in combination with heterologous nucleic acids. It also extends tothe genomic DNA or cDNA or part thereof encoding F3′H or part thereof inreverse orientation relative to its or another promoter. It furtherextends to naturally-occurring sequences following at least a partialpurification relative to other nucleic acid sequences.

The term “nucleic acid molecule” includes a nucleic acid isolate and agenetic sequence and is used herein in its most general sense andencompasses any contiguous series of nucleotide bases specifyingdirectly, or via a complementary series of bases, a sequence of aminoacids in a F3′H. Such a sequence of amino acids may constitute afull-length F3′H or an active truncated form thereof or may correspondto a particular region such as an N-terminal, C-terminal or internalportion of the enzyme. The nucleic acid molecules contemplated hereinalso encompass oligonucleotides useful as genetic probes or as“antisense” molecules capable of regulating expression of thecorresponding gene in a plant. An “antisense molecule” as used hereinmay also encompass a gene construct comprising the structural genomic orcDNA gene or part thereof in reverse orientation relative to its own oranother promoter. Accordingly, the nucleic acid molecules of the presentinvention may be suitable for use as cosuppression molecules, ribozymemolecules, sense molecules and antisense molecules to modulate levels of3′-hydroxylated anthocyanins.

In one embodiment, the nucleic acid molecule encoding F3′H or variousderivatives thereof is used to reduce the activity of an endogenousF3′H, or alternatively the nucleic acid molecule encoding this enzyme orvarious derivatives thereof is used in the antisense orientation toreduce activity of the F3′H. Although not wishing to limit the presentinvention to any one theory, it is possible that the introduction of thenucleic acid molecule into a cell results in this outcome either bydecreasing transcription of the homologous endogenous gene or byincreasing turnover of the corresponding mRNA. This may be achievedusing gene constructs containing F3′H nucleic acid molecules or variousderivatives thereof in either the sense or the antisense orientation. Ina further alternative, ribozymes could be used to inactivate targetnucleic acid molecules. Alternatively, the nucleic acid molecule encodesa functional F3′H and this is used to elevate levels of this enzyme inplants.

Reference herein to the altering of flavonoid F3′H activity relates toan elevation or reduction in activity of up to 30% or more preferably of30-50%, or even more preferably 50-75% or still more preferably 75% orgreater above or below the normal endogenous or existing levels ofactivity. The level of activity can be readily assayed using a modifiedversion of the method described by Stotz and Forkmann (1982) (seeExample 7) or by assaying for the amount of F3′H product such asquercetin, cyanidin or peonidin as set forth in Example 5.

The present invention further extends to nucleic acid molecules in theform of oligonucleotide primers or probes capable of hybridizing to aportion of the nucleic acid molecules contemplated above, and inparticular those selected from the nucleic acid molecules set forth inSEQ ID NOs: 1, 3, 5, 7, 9, 14, 16, 18, 20, 22 or 24 under high,preferably under medium and most preferably under low stringencyconditions. Preferably the portion corresponds to the 5′ or the 3′ endof the F3′H gene. For convenience the 5′ end is considered herein todefine a region substantially between the 5′ end of the primarytranscript to a centre portion of the gene, and the 3′ end is consideredherein to define a region substantially between the centre portion ofthe gene and the 3′ end of the primary transcript. It is clear,therefore, that oligonucleotides or probes may hybridize to the 5′ endor the 3′ end or to a region common to both the 5′ and the 3′ ends.

The nucleic acid molecule or its complementary form may encode thefull-length enzyme or a part or derivative thereof. By “derivative” ismeant any single or multiple amino acid substitutions, deletions, and/oradditions relative to the naturally-occurring enzyme and includes parts,fragments, portions, fusion molecules, homologues and analogues. In thisregard, the nucleic acid includes the naturally-occurring nucleotidesequence encoding F3′H or may contain single or multiple nucleotidesubstitutions, deletions and/or additions to said naturally-occurringsequence. A fusion molecule may be a fusion between nucleotide sequencesencoding two or more F3′Hs, or a fusion between a nucleotide sequenceencoding an F3′H and a nucleotide sequence encoding any otherproteinaceous molecule. Fusion molecules are useful in alteringsubstrate specificity.

A derivative of the nucleic acid molecule or its complementary form, orof a F3′H, of the present invention may also include a “part”, whetheractive or inactive. An active or functional nucleic acid molecule is onewhich encodes an enzyme with F3′H activity. An active or functionalmolecule further encompasses a partially-active molecule; for example,an F3′H with reduced substrate specificity would be regarded aspartially active. A derivative of a nucleic acid molecule may be usefulas an oligonucleotide probe, as a primer for polymerase chain reactionsor in various mutagenic techniques, for the generation of antisensemolecules or in the construction of ribozymes. They may also be usefulin developing co-suppression constructs. The nucleic acid moleculeaccording to this aspect of the present invention may or may not encodea functional F3′H. A “part” may be derived from the 5′ end or the 3′ endor a region common to both the 5′ and the 3′ ends of the nucleic acidmolecule.

Amino acid insertional derivatives of the F3′H of the present inventioninclude amino and/or carboxyl terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Insertional amino acidsequence variants are those in which one or more amino acid residues areintroduced into a predetermined site in the protein although randominsertion is also possible with suitable screening of the resultingproduct. Deletional variants are characterised by the removal of one ormore amino acids from the sequence. Substitutional amino acid variantsare those in which at least one residue in the sequence has been removedand a different residue inserted in its place. Typical substitutions arethose made in accordance with Table 1 below. TABLE 1 Suitable residuesfor amino acid substitutions Original Residue Exemplary SubstitutionsAla Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile PheMet; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Where the F3′H is derivatised by amino acid substitution, the aminoacids are generally replaced by other amino acids having likeproperties, such as hydrophobicity, hydrophilicity, electronegativity,bulky side chains and the like. Amino acid substitutions are typicallyof single residues. Amino acid insertions will usually be in the orderof about 1-10 amino acid residues and deletions will range from about1-20 residues. Preferably, deletions or insertions are made in adjacentpairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made usingpeptide synthetic techniques well known in the art, such as solid phasepeptide synthesis (Merrifield, 1964) and the like, or by recombinant DNAmanipulations. Techniques for making substitution mutations atpredetermined sites in DNA having known or partially known sequence arewell known and include, for example, M13 mutagenesis. The manipulationof DNA sequence to produce variant proteins which manifest assubstitutional, insertional or deletional variants are convenientlydescribed, for example, in Sambrook et al. (1989).

Other examples of recombinant or synthetic mutants and derivatives ofthe F3′H of the present invention include single or multiplesubstitutions, deletions and/or additions of any molecule associatedwith the enzyme such as carbohydrates, lipids and/or proteins orpolypeptides.

The terms “analogues” and “derivatives” also extend to any chemicalequivalents of the F3′H, whether functional or not, and also to anyamino acid derivative described above. Where the “analogues” and“derivatives” of this aspect of the present invention arenon-functional, they may act as agonists or antagonists of F3′Hactivity. For convenience, reference to “F3′H” herein includes referenceto any derivatives, including parts, mutants, fragments, homologues oranalogues thereof.

The present invention is exemplified using nucleic acid sequencesderived from petunia, carnation, rose, snapdragon, arabidopsis,chrysanthemum, lisianthus, torenia, morning glory and gentian, sincethese represent the most convenient and preferred sources of material todate. However, one skilled in the art will immediately appreciate thatsimilar sequences can be isolated from any number of sources such asother plants or certain microorganisms. Examples of other plantsinclude, but are not limited to, maize, tobacco, cornflower,pelargonium, apple, gerbera and african violet. All such nucleic acidsequences encoding directly or indirectly a flavonoid pathway enzyme andin particular F3′H, regardless of their source, are encompassed by thepresent invention.

The nucleic acid molecules contemplated herein may exist in eitherorientation alone or in combination with a vector molecule, for examplean expression-vector. The term vector molecule is used in its broadestsense to include any intermediate vehicle for the nucleic acid molecule,capable of facilitating transfer of the nucleic acid into the plant celland/or facilitating integration into the plant genome. An intermediatevehicle may, for example, be adapted for use in electroporation,microprojectile bombardment, Agrobacterium-mediated transfer orinsertion via DNA or RNA viruses. The intermediate vehicle and/or thenucleic acid molecule contained therein may or may not need to be stablyintegrated into the plant genome. Such vector molecules may alsoreplicate and/or express in prokaryotic cells. Preferably, the vectormolecules or parts thereof are capable of integration into the plantgenome. The nucleic acid molecule may additionally contain a promotersequence capable of directing expression of the nucleic acid molecule ina plant cell. The nucleic acid molecule and promoter may also beintroduced into the cell by any number of means such as those describedabove.

In accordance with the present invention, a nucleic acid moleculeencoding a F3′H or a derivative or part thereof may be introduced into aplant in either orientation to allow, permit or otherwise facilitatemanipulation of levels of production of mRNA in the cytoplasm and/orproduction of enzyme from the mRNA, thereby providing a means either toconvert DHK and/or other suitable substrates, if synthesised in theplant cell, ultimately into anthocyanin derivatives of anthocyanidinssuch as cyanidin and/or peonidin; or alternatively to inhibit suchconversion of metabolites by reducing or eliminating endogenous orexisting F3′H activity. The production of mRNA in the cytoplasm and/orproduction of enzyme from the mRNA, is referred to herein as“expression”. The production of anthocyanins contributes to theproduction of a red or blue flower colour. Expression of the nucleicacid molecule in either orientation in the plant may be constitutive,inducible or developmental, and may also be tissue-specific.

According to this aspect of the present invention there is provided amethod for producing a transgenic plant capable of synthesizing F3′H orfunctional derivatives thereof, said method comprising stablytransforming a cell of a suitable plant with a nucleic acid moleculewhich comprises a sequence of nucleotides encoding said F3′H, underconditions permitting the eventual expression of said nucleic acidmolecule, regenerating a transgenic plant from the cell and growing saidtransgenic plant for a time and under conditions sufficient to permitthe expression of the nucleic acid molecule. The transgenic plant maythereby produce elevated levels of F3′H activity relative to the amountexpressed in a comparable non-transgenic plant.

Another aspect of the present invention contemplates a method forproducing a transgenic plant with reduced endogenous or existing F3′Hactivity, said method comprising stably transforming a cell of asuitable plant with a nucleic acid molecule which comprises a sequenceof nucleotides encoding or complementary to a sequence encoding F3′H,regenerating a transgenic plant from the cell and where necessarygrowing said transgenic plant under conditions sufficient to permit theexpression of the nucleic acid molecule.

Yet another aspect of the present invention contemplates a method forproducing a genetically modified plant with reduced endogenous orexisting F3′H activity, said method comprising altering the F3′H genethrough modification of the endogenous sequences via homologousrecombination from an appropriately altered F3′H gene or derivative orpart thereof introduced into the plant cell, and regenerating thegenetically modified plant from the cell.

In accordance with these aspects of the present invention the preferrednucleic acid molecules are substantially as set forth in SEQ ID NO: 1,3, 5, 7, 14, 16, 18, 20, 22, 24, or the coding region of 9, or have atleast about 60% similarity thereto, or are capable of hybridisingthereto under low stringency conditions.

In a preferred embodiment, the present invention contemplates a methodfor producing a transgenic flowering plant exhibiting altered flowercolour, said method comprising stably transforming a cell of a suitableplant with a nucleic acid molecule of the present invention,regenerating a transgenic plant from the cell and growing saidtransgenic plant for a time and under conditions sufficient to permitthe expression of the nucleic acid molecule into the F3′H enzyme.Alternatively, said method may comprise stably transforming a cell of asuitable plant with a nucleic acid molecule of the present invention orits complementary sequence, regenerating a transgenic plant from thecell and growing said transgenic plant for a time and under conditionssufficient to alter the level of activity of the endogenous or existingF3′H. Preferably, the altered level would be less than the endogenous orexisting level of F3′H activity in a comparable non-transgenic plant.

In a related embodiment, the present invention contemplates a method forproducing a flowering plant exhibiting altered flower colour, saidmethod comprising alteration of the F3′H gene through modification ofthe endogenous sequences via homologous recombination from anappropriately altered F3′H gene or derivative thereof introduced intothe plant cell and regenerating the genetically modified plant from thecell.

The nucleic acid molecules of the present invention may or may not bedevelopmentally regulated. Preferably, the modulation of levels of3′-hydroxylated anthocyanins leads to altered flower colour whichincludes the production of red flowers or other colour shades dependingon the physiological conditions of the recipient plant. By ‘recipientplant’ is meant a plant capable of producing a substrate for the F3′Henzyme, or producing the F3′H enzyme itself, and possessing theappropriate physiological properties and genotype required for thedevelopment of the colour desired. This may include but is not limitedto petunia, carnation, chrysanthemum, rose, snapdragon, tobacco,cornflower, pelargonium, lisianthus, gerbera, apple, iris, lily, africanviolet, gentian, torenia and Japanese morning glory.

Accordingly, the present invention extends to a method for producing atransgenic plant capable of modulating levels of 3′-hydroxylatedanthocyanins, said method comprising stably transforming a cell or groupof cells of a suitable plant with a nucleic acid molecule comprising asequence of nucleotides encoding, or complementary to a sequenceencoding, F3′H or a derivative thereof, and regenerating a transgenicplant from said cell or cells.

One skilled in the art will immediately recognise the variationsapplicable to the methods of the present invention, such as increasingor decreasing the level of enzyme activity of the enzyme naturallypresent in a target plant leading to differing shades of colours.

The present invention, therefore, extends to all transgenic plantscontaining all or part of the nucleic acid module of the presentinvention and/or any homologues or related forms thereof or antisenseforms of any of these and in particular those transgenic plants whichexhibit altered flower colour. The transgenic plants may contain anintroduced nucleic acid molecule comprising a nucleotide sequenceencoding or complementary to a sequence encoding F3′H. Generally, thenucleic acid would be stably introduced into the plant genome, althoughthe present invention also extends to the introduction of the F3′Hnucleotide sequence within an autonomously-replicating nucleic acidsequence such as a DNA or RNA virus capable of replicating within theplant cell. The invention also extends to seeds from such transgenicplants. Such seeds, especially if coloured, will be useful asproprietary tags for plants.

A further aspect of the present invention is directed to recombinantforms of F3′H. The recombinant forms of the enzymes will provide asource of material for research to develop, for example, more activeenzymes and may be useful in developing in vitro systems for productionof coloured compounds.

Still a further aspect of the present invention contemplates the use ofthe genetic sequences described herein in the manufacture of a geneticconstruct capable of use in modulating levels of 3′-hydroxylatedanthocyanins in a plant or cells of a plant.

Yet a further aspect of the present invention provides flowers and inparticular cut flowers, from the transgenic plants herein described,exhibiting altered flower colour.

Another aspect of the present invention is directed to a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding, a F3′H or a derivative thereof wherein saidnucleic acid molecule is capable of being expressed in a plant cell. Theterm “expressed” is equivalent to the term “expression” as definedabove.

The nucleic acid molecules according to this and other aspects of theinvention allow, permit or otherwise facilitate increased efficiency inmodulation of 3′-hydroxylated anthocyanins relative to the efficency ofthe pCGP619 cDNA insert contained in plasmid pCGP809, disclosed inInternational Patent Application No. PCT/AU93/00127 [WO 93/20206]. Theterm “plant cell” includes a single plant cell or a group of plant cellssuch as in a callus, plantlet or plant or parts thereof includingflowers and seeds.

Another aspect of the present invention provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence of nucleotides encoding a F3′H, wherein the translation of thesaid nucleic acid molecule comprises the amino acid sequence RPPNSGA.Preferably, the translation of the said nucleic acid molecule comprisesthe amino acid sequence RPPNSGAXHXAYNYXDL and still more preferably thetranslation of the said nucleic acid molecule comprises the amino acidsequence RPPNSGAXHXAYNYXDL[X]_(n)GGEK, where X represents any amino acidand [X]_(n) represents an amino acid sequence of from 0 to 500 aminoacids.

The present invention is further described by reference to the followingnon-limiting Figures and Examples.

In the Figures:

FIGS. 1 a and 1 b are schematic representations of the flavonoidbiosynthesis pathways in P. hybrida flowers showing the enzymes andgenetic loci involved in the conversions. Enzymes involved in thepathway have been indicated as follows: PAL=phenylalanine ammonia-lyase;C4H=cinnamate 4-hydroxylase; 4CL=4-coumarate: CoA ligase; CHS=chalconesynthase; CHI=chalcone isomerase; F3H=flavanone 3-hydroxylase;F3′H=flavonoid 3′-hydroxylase; F3′5′ H=flavonoid 3′5′ hydroxylase;FLS=flavonol synthase; DFR=dihydroflavonol-4-reductase; ANS=anthocyaninsynthase; 3GT=UDP-glucose: anthocyanin-3-glucoside; 3RT=UDP-rhamnose:anthocyanidin-3-glucoside rhamnosyltransferase;ACT=anthocyanidin-3-rutinoside acyltransferase; 5GT=UDP-glucose:anthocyanin 5-glucosyltransferase; 3′ OMT=anthocyaninO-methyltransferase; 3′, 5′ OMT=anthocyanin 3′, 5′ O-methyltransferase.Three flavonoids in the pathway are indicated as:P-3-G=pelargonidin-3-glucoside; DHM=dihydomyricetin;DHQ=dihydroquercetin. The flavonol, myricetin, is only produced at lowlevels and the anthocyanin, pelargonidin, is rarely produced in P.hybrida.

FIG. 2 is a diagrammatic representation of the plasmid pCGP161containing a cDNA clone (F1) representing the cinnamate-4-hydroxylasefrom P. hybrida. ³²P-labelled fragments of the 0.7 kb EcoRI/XhoIfragment were used to probe the Old Glory Red petal cDNA library. Fordetails, refer to Example 4. Abbreviations are as follows: Amp=theampicillin resistance gene; ori=origin of replication; T3=recognitionsequence for T3 RNA polymerase; T7=recognition sequence for T7 RNApolymerase. Restriction enzyme sites are also marked.

FIG. 3 is a diagrammatic representation of the plasmid pCGP602containing a cDNA clone (617) representing a flavonoid 3′5′ hydroxylase(Hf1) from P. hybrida. ³²P-labelled fragments of the 1.6 kb BspHI/FspIfragment containing the Hf1 coding region were used to probe the OldGlory Red petal cDNA library. For details, refer to Example 4.Abbreviations are as follows: Amp=the ampicillin resistance gene;ori=origin of replication; T3=recognition sequence for T3 RNApolymerase; T7=recognition sequence for T7 RNA polymerase. Restrictionenzyme sites are also marked.

FIG. 4 is a diagrammatic representation of the plasmid pCGP175containing a cDNA clone (H2) representing a flavonoid 3′5′ hydroxylase(Hf2) from P. hybrida. ³²P-labelled fragments of the 1.3 kb EcoRI/XhoIand 0.5 kb XhoI fragments which together contain the Hf2 coding regionwere used to probe the Old Glory Red petal cDNA library. For details,refer to Example 4. Abbreviations are as follows: Amp=the ampicillinresistance gene; ori=origin of replication; T3=recognition sequence forT3 RNA polymerase; T7=recognition sequence for T7 RNA polymerase.Restriction enzyme sites are also marked.

FIG. 5 is a diagrammatic representation of the plasmid pCGP619containing the 651 cDNA clone representing a cytochrome P450 from P.hybrida. ³²P-labelled fragments of the 1.8 kb EcoRI/XhoI fragment wereused to probe the Old Glory Red petal cDNA library. For details, referto Example 4. Abbreviations are as follows: Amp=the ampicillinresistance gene; ori=origin of replication; T3=recognition sequence forT3 RNA polymerase; T7=recognition sequence for T7 RNA polymerase.Restriction enzyme sites are also marked.

FIG. 6 is a representation of an autoradiograph of an RNA blot probedwith ³²P-labelled fragments of the OGR-38 cDNA clone contained inpCGP1805 (see Example 6). Each lane contained a 20 μg sample of totalRNA isolated from the flowers or leaves of plants of a V23 (ht1/ht1)×VR(Ht1/ht1) backcross population. A 1.8 kb transcript was detected in theVR-like (ht1/ht1) flowers that contained high levels of quercetin(Q+)(lanes 9-14). The same size transcript was detected at much lowerlevels in the V23-like (ht1/ht1) flowers that contained little or noquercetin (Q−) (lanes 3-8). A reduced level of transcript was alsodetected in VR leaves (lane 1) and V23 petals (lane 2). This isdescribed in Example 5.

FIG. 7 is a diagrammatic representation of the yeast expression plasmidpCGP1646 (see Example 7). The OGR-38 cDNA insert from pCGP1805 wascloned in a ‘sense’ orientation behind the yeastglyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) in theexpression vector pYE22m. TRP1=Trp1 gene, IR1=inverted repeat of 2 μmplasmid, TGAP=terminator sequence from the yeastglyceraldehyde-3-phosphate dehydrogenase gene. Restriction enzyme sitesare also marked.

FIG. 8 is a diagrammatic representation of the binary plasmid pCGP1867(described in Example 8). The Ht1 cDNA insert (OGR-38) from pCGP1805 wascloned in a “sense” orientation behind the Mac promoter in theexpression vector of pCGP293. Abbreviations are as follows: LB=leftborder; RB=right border; Gm=the gentamycin resistance gene; 35S=thepromoter region from the Cauliflower Mosaic Virus 35S gene; nptII=theneomycin phosphotransferase II gene; tml3′=the terminator region fromthe tml gene of Agrobacterium; mas3′=the terminator region from themannopine synthase gene of Agrobacterium; ori pRi=a broad host rangeorigin of replication from an Agrobacterium rhizogenes plasmid;oriColE1=a high copy origin of replication from a Colcinin E1 plasmid.Restriction enzyme sites are also marked.

FIG. 9 is a diagrammatic representation of the binary plasmid pCGP1810,preparation of which is described in Example 13. The KC-1 cDNA insertfrom pCGP1807 (see Example 12) was cloned in a “sense” orientationbehind the Mac promoter in the expression vector of pCGP293.Abbreviations are as follows: LB=left border; RB=right border; Gm=thegentamycin resistance gene; 35S=the promoter region from the CauliflowerMosaic Virus 35S gene; nptII=the neomycin phosphotransferase II gene;tml3′=the terminator region from the tml gene of Agrobacterium;mas3′=the terminator region from the mannopine synthase gene ofAgrobacterium; ori pRi=a broad host range origin of replication from aplasmid from Agrobacterium rhizogenes; oriColE1=a high copy origin ofreplication from a Colcinin E1 plasmid. Restriction enzyme sites arealso marked.

FIG. 10 is a diagrammatic representation of the binary plasmid pCGP1813,construction of which is described in Example 14. The KC-1 cDNA insertfrom pCGP1807 (see Example 12) was cloned in a “sense” orientationbetween the mac promoter and mas terminator. The Mac: KC-1: masexpression cassette was subsequently cloned into the binary vectorpWTT2132. Abbreviations are as follows: Tet=the tetracycline resistancegene; LB=left border; RB=right border, surB=the coding region andterminator sequence from the acetolactate synthase gene; 35S=thepromoter region from the cauliflower mosaic virus 35S gene, mas3′=theterminator region from the mannopine synthase gene of Agrobacterium;pVS1=a broad host range origin of replication from a plasmid fromPseodomonas aeruginosa, pACYCori=modified replicon from pACYC184 from E.coli. Restriction enzyme sites are also marked.

FIG. 11 is a representation of an autoradiograph of a Southern blotprobed with ³²P labelled fragments of the Am3Ga differential display PCRfragment (as described in Example 16). Each lane contained a 10 μgsample of EcoRV-digested genomic DNA isolated from N8 (Eos⁺), K16 (eos⁻)or plants of an K16×N8 F₂ population. Hybridizing bands were detected inthe genomic DNA from cyanidin-producing plants (indicated with “+”)(Lanes 1, 3, 4, 5, 6, 7, 9, 10, 12 and 15). No specific hybridizationwas observed in the genomic DNA samples from non-cyanidin-producingplants (indicated with “−”) (Lanes 2, 8, 11, 13 and 14).

FIG. 12 is a representation of an autoradiograph of an RNA blot probedwith ³²P-labelled fragments of the Am3Ga differential display PCRfragment. Each lane contained a 10 μg sample of total RNA isolated fromthe flowers or leaves of plants of an N8 (Eos⁺)×K16 (eos⁻) F₂population. A 1.8 kb transcript was detected in the K16×N8 F₂ flowersthat produced cyanidin (cyanidin +) (plants #1, #3, #4, #5 and #8). Notranscript was detected in the K16×N8 F₂ flowers that did not producecyanidin (cyanidin −) (plants #6, #11, #12) or in a leaf sample (#13L)from an K16×N8 F₂ plant that produced cyanidin in the flowers. Detailsare provided in Example 17.

FIG. 13 is a diagrammatic representation of the binary plasmid pCGP250,construction of which is described in Example 20. The sdF3′H cDNAinsert, containing the nucleotides 1 through to 1711 (SEQ ID NO:5) frompCGP246 (see Example 18), was cloned in a “sense” orientation behind theMac promoter in the expression vector of pCGP293. Abbreviations are asfollows: LB=left border; RB=right border; Gm=the gentamycin resistancegene; 35S=the promoter region from the Cauliflower Mosaic Virus 35Sgene; nptII=the neomycin phosphotransferase II gene; tml3′=theterminator region from the tml gene of Agrobacterium; mas3′=theterminator region from the mannopine synthase gene of Agrobacterium; oripRi=a broad host range origin of replication from a plasmid fromAgrobacterium rhizogenes; oriColE1=a high copy origin of replicationfrom a Colcinin E1 plasmid. Restriction enzyme sites are also marked.

FIG. 14 is a diagrammatic representation of the binary plasmid pCGP231,construction of which is described in Example 20. The sdF3′H cDNAinsert, containing the nucleotides 104 through to 1711 (SEQ ID NO:5)from pCGP246, was cloned in a “sense” orientation behind the Macpromoter in the expression vector of pCGP293. Abbreviations are asfollows: LB=left border; RB=right border; Gm=the gentamycin resistancegene; 35S=the promoter region from the Cauliflower Mosaic Virus 35Sgene; nptII=the neomycin phosphotransferase II gene; tml3′=theterminator region from the tml gene of Agrobacterium; mas3′=theterminator region from the mannopine synthase gene of Agrobacterium; oripRi=a broad host range origin of replication from a plasmid fromAgrobacterium rhizogenes; oriColE1=a high copy origin of replicationfrom a Colcium E1 plasmid. Restriction enzyme sites are also marked.

FIG. 15 is a diagrammatic representation of the binary plasmidpBI-Tt7-2. The 6.5 kb EcoRI/SalI Tt7 genomic fragment from E-5 wascloned into EcoRI/SalI-cut pBI101, replacing the resident GUS gene. Theorientation of the Tt7 (F3′H) gene as indicated (5′ to 3′) wasdetermined through DNA sequencing. Abbreviations are as follows: LB=leftborder; RB=right border; nos 5′=the promoter region from the nopalinesynthase gene of Agrobacterium; nptII=the coding region of the neomycinphosphotransferase II gene; nos 3′=the terminator region from thenopaline synthase gene of Agrobacterium; nptI=the coding region of theneomycin phosphotransferase I gene. Restriction enzyme sites are alsomarked.

FIG. 16 is a diagrammatic representation of the binary plasmid pCGP2166,construction of which is described in Example 26. The rose #34 cDNAinsert from pCGP2158 (see Example 25) was cloned in a “sense”orientation behind the Mac promoter in the expression vector of pCGP293.Abbreviations are as follows: LB=left border; RB=right border; Gm=thegentamycin resistance gene; 35S=the promoter region from the CauliflowerMosaic Virus 35S gene; nptII=the neomycin phosphotransferase II gene;tml3′=the terminator region from the tml gene of Agrobacterium;mas3′=the terminator region from the mannopine synthase gene ofAgrobacterium; ori pRi=a broad host range origin of replication from aplasmid from Agrobacterium rhizogenes; oriColE1=a high copy origin ofreplication from a Colcinin E1 plasmid. Restriction enzyme sites arealso marked.

FIG. 17 is a diagrammatic representation of the binary plasmid pCGP2169construction of which is described in Example 27. The rose #34 cDNAinsert from pCGP2158 was cloned in a “sense” orientation between theCaMV35S promoter and the ocs terminator. The 35S: rose #34: ocsexpression cassette was subsequently cloned into the binary vectorpWM2132. Abbreviations are as follows: Tet=the tetracycline resistancegene; LB=left border; RB right border; surB=the boding region andterminator sequence from the acetolactate synthase gene; 35S=thepromoter region from the cauliflower mosaic virus 35S gene,ocs=terminator region from the octopine synthase gene fromAgrobacterium; pVS1=a broad host range origin of replication from aplasmid from Pseodomous aeruginosa, pACYCori=modified replicon frompACYC184 from E. coli. Restriction enzyme sites are also marked.

FIG. 18 is a diagrammatic representation of the binary plasmid pLN85,construction of which is described in Example 28. The chrysanthemum RM6icDNA insert from pCHRM1 was cloned in “anti-sense” orientation behindthe promoter from the Cauliflower Mosaic Virus 35S gene (35S). Otherabbreviations are as follows: LB=left border; RB=right border; ocs3′=theterminator region from the octopine synthase gene of Agrobacterium;pnos:nptII:nos 3′=the expression cassette containing the promoter regionfrom the nopaline synthase gene of Agrobacterium; the coding region ofthe neomycin phosphotransferase II gene and the terminator region fromthe nopaline synthase gene of Agrobacterium; oriT=origin of transfer ofreplication; trfA*=a trans-acting replication function; oriColE1=a highcopy origin of replication from a Colcinin E1 plasmid; Tn7SpR/StR=thespectinomycin and streptomycin resistance genes from transposon Tn7;oriVRK2=a broad host range origin of replication from plasmid RK2.Restriction enzyme sites are also marked.

FIG. 19 is a diagrammatic representation of the yeast expression plasmidpYTHT6, construction of which is described in Example 30. The THT6 cDNAinsert from pTHT6 was cloned in a “sense” orientation behind the yeastglyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) in theexpression vector pYE22m. Abbreviations are as follows: TRP1=Trp1 gene;IR1=inverted repeat of 2 μm plasmid; TGAP=the terminator sequence fromthe yeast glyceraldehyde-3-phosphate dehydrogenase gene. Restrictionenzyme sites are also marked.

Amino acid abbreviations used throughout the specification are shown inTable 2, below. TABLE 2 Amino acid abbreviations Amino acid 3-lettersingle-letter L-alanine Ala A L-arginine Arg R L-asparagine Asn NL-aspartic acid Asp D L-cysteine Cys C L-glutamine Gln Q L-glutamic acidGlu E L-glycine Gly G L-histidine His H L-isoleucine Ile I L-leucine LeuL L-lysine Lys K L-methionine Met M L-phenylalanine Phe F L-proline ProP L-serine Ser S L-threonine Thr T L-tryptophan Trp W L-tyrosine Tyr YL-valine Val V

Table 3 provides a summary of the SEQ ID NO's assigned to the sequencesreferred to herein: TABLE 3 Sequence Species SEQ ID NO cDNA insert ofpCGP1805 Petunia SEQ ID NO: 1 corresponding amino acid sequence PetuniaSEQ ID NO: 2 cDNA insert of pCGP1807 Carnation SEQ ID NO: 3corresponding amino acid sequence Carnation SEQ ID NO: 4 cDNA insert ofpCGP246 Snapdragon SEQ ID NO: 5 corresponding amino acid sequenceSnapdragon SEQ ID NO: 6 cDNA partial sequence Arabidopsis SEQ ID NO: 7corresponding amino acid sequence Arabidopsis SEQ ID NO: 8 genomicsequence Arabidopsis SEQ ID NO: 9 corresponding amino acid sequenceArabidopsis SEQ ID NO: 10 for exon I corresponding amino acid sequenceArabidopsis SEQ ID NO: 11 for exon II corresponding amino acid sequenceArabidopsis SEQ ID NO: 12 for exon III corresponding amino acid sequenceArabidopsis SEQ ID NO: 13 for exon IV cDNA insert of pCGP2158 Rose SEQID NO: 14 corresponding amino acid sequence Rose SEQ ID NO: 15 cDNAinsert of pCHRM1 Chrysan- SEQ ID NO: 16 themum corresponding amino acidsequence Chrysan- SEQ ID NO: 17 themum THT cDNA sequence Torenia SEQ IDNO: 18 corresponding amino acid sequence Torenia SEQ ID NO: 19 MHT 85cDNA sequence Jap. Morning SEQ ID NO: 20 Glory corresponding amino acidsequence Jap. Morning SEQ ID NO: 21 Glory GHT13 cDNA sequence GentianSEQ ID NO: 22 corresponding amino acid sequence Gentian SEQ ID NO: 23cDNA insert of pL3-6 Lisianthus SEQ ID NO: 24 corresponding amino acidsequence Lisianthus SEQ ID NO: 25 cDNA sequence from WO 93/20206 PetuniaSEQ ID NO: 26 oligonucleotide polyT-anchA SEQ ID NO: 27 oligonucleotidepolyT-anchC SEQ ID NO: 28 oligonucleotide polyT-anchG SEQ ID NO: 29conserved amino acid primer region SEQ ID NO: 30 correspondingoligonucleotide sequence SEQ ID NO: 31 conserved amino acid primerregion SEQ ID NO: 32 corresponding oligonucleotide sequence SEQ ID NO:33 oligonucleotide primer Pet Haem-New SEQ ID NO: 34 conserved aminoacid primer region SEQ ID NO: 35 corresponding oligonucleotide sequenceSEQ ID NO: 36 oligonucleotide Snapred Race A SEQ ID NO: 37oligonucleotide Snapred Race C SEQ ID NO: 38 oligonucleotide poly-C RaceSEQ ID NO: 39 oligonucleotide primer Pet Haem SEQ ID NO: 40

The disarmed microorganism Agrobacterium tumefaciens strain AGL0separately containing the plasmids pCGP1867, pCGP1810 and pCGP231 weredeposited with the Australian Government Analytical Laboratories, 1Suakin Street, Pymble, New South Wales, 2037, Australia on 23 Feb., 1996and were given Accession Numbers 96/10967, 96/10968 and 96/10969,respectively.

Isolation of Flavonoid 3′-Hydroxylase and Related Nucleic Acid SequencesEXAMPLE 1 Plant Material

Petunia

The Petunia hybrida varieties used are presented in Table 4. TABLE 4Plant variety Properties Source/Reference Old Glory F₁ Hybrid Ball Seed,USA Blue (OGB) Old Glory F₁ Hybrid Ball Seed, USA Red (OGR) V23 An1,An2, An3, An4, An6, An8, Wallroth et al. (1986) An9, An10, ph1, Hf1,Hf2, ht1, Doodeman et al. (1984) Rt, po, Bl, Fl R51 An1, An2, An3, an4,An6, An8, Wallroth et al. (1986) An9, An10, An11, Ph1, hf1, hf2,Doodeman et al. (1984) Ht1, rt, Po, bl, fl VR V23 × R51 F₁ Hybrid SW63An1, An2, An3, an4, An6, An8, I.N.R.A., Dijon, Cedex An9, An10, An11,Ph1, Ph2, Ph5, France hf1, hf2, ht1, ht2, po, mf1, fl Skr4 An1, An2,An3, An4, An6, An11, I.N.R.A., Dijon, Cedex hf1, hf2, ht1, Ph1, Ph2,Ph5, rt, France Po, Mf1, Mf2, fl Skr4 × F₁ Hybrid SW63

Plants were grown in specialised growth rooms with a 14 hour day lengthat a light intensity of 10,000 lux and a temperature of 22° C. to 26° C.

Carnation

Flowers of Dianthus caryophyllus cv. Kortina Chanel were obtained fromVan Wyk and Son Flower Supply, Victoria.

Dianthus caryophyllus flowers were harvested at developmental stagesdefined as follows:

-   Stage 1: Closed bud, petals not visible.-   Stage 2: Flower buds opening: tips of petals visible.-   Stage 3: Tips of nearly all petals exposed. “Paint-brush stage”.-   Stage 4: Outer petals at 45° angle to stem.-   Stage 5: Flower fully open.    Snapdragon

The Antirrhinum majus lines used were derived from the parental linesK16 (eos⁻) and N8 (Eos⁺). A strict correlation exists between F3′Hactivity and the Eos gene which is known to control the 3′-hydroxylationof flavones, flavonols and anthocyanins (Forkmann and Stotz, 1981). K16is a homozygous recessive mutant lacking F3′H activity while N8 is wildtype for F3′H activity. These lines are similar, though not isogenic.Both parental lines and the seed from a selfed (K16×N8) F₁ plant wereobtained from Dr C. Martin (John Innes Centre, Norwich, UK).

Arabidopsis

The Arabidopsis thaliana lines Columbia Tt7), Landsberg erecta (Tt7) andNW88 (tt7) were obtained from the Nottingham Arabidopsis Stock Centre.Wild-type A. thaliana (TL) seeds have a characteristic brown colour.Seeds of u mutants have pale brown seeds and the plants arecharacterized by a reduced anthocyanin content in leaves (Koornneef etal., 1982). Tt7 plants produce cyanidin while tt7 mutants accumulatepelargonidin, indicating that the Tt7 gene controls flavonoid3′-hydroxylation.

Rose

Flowers of Rosa hybrida cv. Kardinal were obtained from Van Wyk and SonFlower Supply, Victoria.

Stages of Rosa hybrida flower development were defined as follows:

-   Stage 1: Unpigmented, tightly closed bud (10-12 mm high; 5 mm wide).-   Stage 2: Pigmented, tightly closed bud (15 mm high; 9 mm wide).-   Stage 3: Pigmented, closed bud; sepals just beginning to open (20-25    mm high; 13-15 mm wide)-   Stage 4: Flower bud beginning to open; petals heavily pigmented;    sepals have separated (bud is 25-30 mm high and 18 mm wide).-   Stage 5: Sepals completely unfolded; some curling. Petals are    heavily pigmented and unfolding (bud is 30-33 mm high and 20 mm    wide).    Chrysanthemum

Stages of Chrysanthemum flower development were defined as follows:

-   Stage 0: No visible flower bud.-   Stage 1: Flower bud visible: florets completely covered by the    bracts.-   Stage 2: Flower buds opening: tips of florets visible.-   Stage 3: Florets tightly overlapped.-   Stage 4: Tips of nearly all florets exposed; outer florets opening    but none horizontal.-   Stage 5: Outer florets horizontal.-   Stage 6: Flower approaching maturity.

EXAMPLE 2 Bacterial Strains

The Escherichia coli strains used were:

-   DH5α sup E44, Δ(lacZYA-ArgF)U169, ø80lacZΔM15, hsdR17 (r_(k)−,    m_(k)+), recA1, end A1, gyrA96, thi-1, relA1, deoR (Hanahan, 1983    and BRL, 1986).-   XL1-Blue MRF′Δ(mcr A)183, Δ(mcrCB-hsdSMR-mrr)173, end A1, sup E44,    thi-1, recA1, gyrA96, relA1, lac[F′ pro AB, lacIqZΔM15, Tn10(Tet    ^(r))]^(C) (Stratagene)-   XL1-Blue supE44, hsdR17 (r_(k)−, m_(k)+), recA1, endA1, gyrA96,    thi-1, relA1, lac[F′ pro AB, lacI^(q), lacZΔM15, Tn10(tet^(r))]-   SOLR e14-(mcrA), Δ(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ,    umuC::Tn5(kan^(r)), uvrC,lac, gyrA96, thi-1, relA1, [F′proAB,    lacI^(q)ZΔM15], Su⁻(non-suppressing) (Stratagene)-   DH10 B(Zip) F-mcrA, Δ(mrr-hsdRMS-mcrBC), ø80d lacZΔM15, ΔlacX74,    deoR, recA1, recA1, araD139, Δ(ara, leu)7697, galU, galKl^(λ), rspL,    nupG-   Y1090r-ΔlacU169, (Δlon)?, araD139, strA, supF, mcrA,    trpC22::Tn10(Tet^(r)) [pMC9 Amp^(r), Tet^(r)], mcrB, hsdR.

The disarmed Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991)was obtained from R. Ludwig (Department of Biology, University ofCalifornia, Santa Cruz, USA).

The cloning vector pBluescript was obtained from Stratagene.

Transformation of the E. coli strain DH5α cells was performed accordingto the method of Inoue et al. (1990).

EXAMPLE 3 General Methods

³²P-Labelling of DNA Probes

DNA fragments (50 to 100 ng) were radioactively labelled with 50 μCi of[α-³²P]-dCTP using an oligolabelling kit (Bresatec). Unincorporated[α-³²P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine)column.

DNA Sequence Analysis

DNA sequencing was performed using the PRISM™ Ready Reaction Dye PrimerCycle Sequencing Kits from Applied Biosystems. The protocols supplied bythe manufacturer were followed. The cycle sequencing reactions wereperformed using a Perkin Elmer PCR machine (GeneAmp PCR System9600) andrun on an automated 373A DNA sequencer (Applied Biosystems).

Homology searches against Genbank, SWISS-PROT and EMBL databases wereperformed using the FASTA and TFASTA programs (Pearson and Lipman, 1988)or BLAST programs (Altschul et al., 1990). Percentage sequencesimilarities were obtained using the LFASTA program (Pearson and Lipman,1988). In all cases ktup values of 6 for nucleotide sequence comparisonsand 2 for amino acid sequence comparisons were used, unless otherwisespecified.

Multiple sequence alignments (ktup value of 2) were performed using theClustalW program incorporated into the MacVector™ 6.0 application(Oxford Molecular Ltd.).

EXAMPLE 4 Isolation of a Flavonoid 3′-hydroxylase (F3′H) cDNA CloneCorresponding to the Ht1 Locus from P. Hybrida cv. Old Glory Red

In order to isolate a cDNA clone that was linked to the Ht1 locus andthat represented the flavonoid 3′-hydroxylase (F3′H) in the petuniaflavonoid pathway, a petal cDNA library was prepared from RNA isolatedfrom stages 1 to 3 of Old Glory Red (OGR) petunia flowers. OGR flowerscontain cyanidin based pigments and have high levels of flavonoid3′-hydroxylase activity. The OGR cDNA library was screened with amixture of ³²P-labelled fragments isolated from three cytochrome P450cDNA clones known to be involved in the flavonoid pathway and from onecytochrome P450 cDNA clone (651) that had flavonoid 3′-hydroxylaseactivity in yeast. These included a petunia cDNA clone representing thecinnamate-4-hydroxylase (C4H) and two petunia cDNA clones (coded by theHf1 and Hf2 loci) representing flavonoid 3′ 5′-hydroxylase (F3′ 5′H)(Holton et al., 1993).

Construction of Petunia cv. OGR cDNA Library

Total RNA was isolated from the petal tissue of P. hybrida cv OGR stage1 to 3 flowers using the method of Turpen and Griffith (1986). Poly(A)⁺RNA was selected from the total RNA, using oligotex-dT™ (Qiagen).

A ZAP-cDNA Gigapack III Gold Cloning kit (Stratagene) was used toconstruct a directional petal cDNA library in λZAP using 5 μg ofpoly(A)+ RNA isolated from stages 1 to 3 of OGR as template. The totalnumber of recombinants obtained was 2.46×10⁶.

After transfecting XL1-Blue MRF′ cells, the packaged cDNA mixture wasplated at 50,000 pfu per 15 cm diameter plate. The plates were incubatedat 37° C. for 8 hours, and the phage were eluted in 100 mM NaCl, 8 mMMgSO₄, 50 mM Tris-HCl pH 8.0, 0.01% (w/v) gelatin (Phage Storage Buffer(PSB)) (Sambrook et al., 1989). Chloroform was added and the phagestored at 4° C. as an amplified library.

100,000 pfu of the amplified library were plated onto NZY plates(Sambrook et al., 1989) at a density of 10,000 pfu per 15 cm plate aftertransfecting XL1-Blue MRF′ cells, and incubated at 37° C. for 8 hours.After incubation at 4° C. overnight, duplicate lifts were taken ontoColony/Plaque Screen™ filters (DuPont) and treated as recommended by themanufacturer.

Isolation of Probes

F3′5′ H Probes

The two flavonoid 3′, 5′ hydroxylases corresponding to the Hf1 or Hf2loci isolated as described in Holton et al. (1993) and U.S. Pat. No.5,349,125, were used in the screening process.

C4H cDNA Clones from Petunia

A number of cytochrome P450 cDNA clones were isolated in the screeningprocess used to loci (Holton et al., 1993; U.S. Pat. No. 5,349,125). Oneof these cDNA clones (F1) (contained in pCGP161) (FIG. 2) was identifiedas representing a cinnamate 4-hydroxylase (C4H), based on sequenceidentity with a previously-characterised C4H clone from mung bean(Mizutani et al., 1993). Sequence data was generated from 295nucleotides at the 5′ end of the petunia F1 cDNA clone. There was 83.1%similarity with the mung bean C4H clone over the 295 nucleotidessequenced and 93.9%-similarity over the predicted amino acid sequence.

651 cDNA Clone

The isolation and identification of the 651 cDNA clone contained inpCGP619 (FIG. 5) was described in the International Patent Application,having publication number WO93/20206. A protein extract of yeastcontaining the 651 cDNA clone under the control of the yeastglyceraldehyde-3-phosphate dehydrogenase promoter of pYE22m (Tanaka etal., 1988) exhibited F3′H activity.

Screening of OGR Library

Prior to hybridization, the duplicate plaque lifts were washed inprewashing solution (50 mM Tris-HCl pH7.5, 1 M NaCl, 1 mM EDTA, 0.1%(w/v) sarcosine) at 65° C. for 30 minutes; stripped in 0.4 M sodiumhydroxide at 65° C. for 30 minutes; then washed in a solution of 0.2 MTris-HCl pH 8.0, 0.1×SSC, 0.1% (w/v) SDS at 65° C. for 30 minutes andfinally rinsed in 2×SSC, 1.0% (w/v) SDS.

The lifts from the OGR cDNA library were screened with ³²P-labelledfragments of (1) a 0.7 kb EcoRI/XhoI fragment from pCGP161 containingthe C4H cDNA clone (FIG. 2), (2) a 1.6 kb BspHI/FspI fragment frompCGP602 containing the Hf1 cDNA clone (FIG. 3), (3) a 1.3 kb XhoIfragment and a 0.5 kb XhoI fragment from pCGP175 containing the codingregion of the Hf2 cDNA clone (FIG. 4) and (4) a 1.8 kb EcoRI/XhoIfragment pCGP619 containing the 651 cDNA clone (FIG. 5).

Hybridization conditions included a prehybridization step in 10% (v/v)formamide, 1 M NaCl, 10% (w/v) dextran sulphate, 1% (w/v) SDS at 42° C.for at least 1 hour. The ³²P_labelled fragments (each at 1×10⁶ cpm/mL)were then added to the hybridization solution and hybridization wascontinued at 42° C. for a further 16 hours. The filters were then washedin 2×SSC, 1% (w/v) SDS at 42° C. for 2×1 hour and exposed to Kodak XARfilm with an intensifying screen at −70° C. for 16 hours.

Two hundred and thirty strongly hybridizing plaques were picked intoPSB. Of these, 39 were rescreened to isolate purified plaques, using thehybridization conditions as described for the initial screening of thecDNA library. The plasmids contained in the λZAP bacteriophage vectorwere rescued and sequence data was generated from the 3′ and 5′ ends ofthe cDNA inserts. Based on sequence homology, 27 of the 39 wereidentical to the petunia cinnamate 4-hydroxylase cDNA clone, 2 of the 39were identical to the Hf1 cDNA clone and 7 of the 39 did not representcytochrome P450s. The remaining 3 cDNA clones (designated as OGR-27,OGR-38, OGR-39) represented “new” cytochrome P450s, compared to thecytochrome P450 clones used in the screening procedure, and were furthercharacterised.

EXAMPLE 5 Restriction Fragment Length Polymorphism (RFLP) Analysis

There are two genetic loci in P. hybrida, Ht1 and Ht2, that controlflavonoid 3′-hydroxylase activity (Tabak et al., 1978; Wiering and deVlaming, 1984). Ht1 is expressed in both the limb and the tube of P.hybrida flowers and gives rise to higher levels of F3′H activity thandoes Ht2 which is only expressed in the tube. The F3′H is able toconvert dihydrokaempferol and naringenin to dihydroquercetin anderiodictyol, respectively. In a flower producing delphinidin-basedpigments, F3′H activity is masked by the F3′5′ H activity. Therefore,the F3′H/F3′5′ H assay (Stotz and Forkmann, 1982) is useless indetermining the presence or absence of F3′H activity. The enzymeflavonol synthase is able to convert dihydrolkaempferol to kaempferoland dihydroquercetin to quercetin (FIG. 1 a). Myricetin, the 3′, 5′hydroxylated flavonol, is produced at low levels in petunia flowers.Therefore, analysing the flowers for the 3′ hydroxylated flavonol,quercetin, infers the presence of F3′H activity.

Restriction Fragment Length Polymorphism (RFLP) analysis of DNA isolatedfrom individual plants in a VR (Ht1/ht1)×V23 (ht1/ht1) backcross wasused to determine which, if any, of the cDNA clones representing P450swere linked to the Ht1 locus. Northern analysis of RNA isolated fromthese plants was also used to detect the presence or absence of atranscript in these lines.

Flowers from a VR (Ht1/ht1)×V23 (ht1/ht1) backcross population wereanalysed for the presence of the flavonols, kaempferol and quercetin. VR(Ht1/ht1) flowers accumulate quercetin and low levels of kaempferolwhile V23 (ht1/ht1) flowers accumulate kaempferol but little or noquercetin. Individual plants from the VR (Ht1/ht1)×V23 (ht1/ht1)backcross were designated as VR-like (Ht1/ht1), if a substantial levelof quercetin was detected in the flower extracts, and V23-like(ht1/ht1), if little or no quercetin but substantial levels ofkaempferol were detected in the flower extracts (see FIG. 6).

Isolation of Genomic DNA

DNA was isolated from leaf tissue essentially as described by Dellaportaet al., (1983). The DNA preparations were further purified by CsClbuoyant density centrifugation (Sambrook et al., 1989).

Southern Blots

The genomic DNA (10 μg) was digested for 16 hours with 60 units of EcoRIand electrophoresed through a 0.7% (w/v) agarose gel in a running bufferof TAE (40 mM Tris-acetate, 50 mM EDTA). The DNA was then denatured indenaturing solution (1.5 M NaCl/0.5 M NaOH) for 1 to 1.5 hours,neutralized in 0.5 M Tris-HCl (pH 7.5)/1.5 M NaCl for 2 to 3 hours andthen transferred to a Hybond N (Amersham) filter in 20×SSC.

RNA Blots

Total RNA was isolated from the petal tissue of P. hybrida cv OGR stage1 to 3 flowers using the method of Turpen and Griffith (1986).

RNA samples were electrophoresed through 2.2 M formaldehyde/1.2% (w/v)agarose gels using running buffer containing 40 mMmorpholinopropanesulphonic acid (pH 7.0), 5 mM sodium acetate, 0.1 mMEDTA (pH 8.0). The RNA was transferred to Hybond-N filters (Amersham) asdescribed by the manufacturer.

Hybridization and Washing Conditions

Southern and RNA blots were probed with ³²P-labelled cDNA fragment (10⁸cpm/μg, 2×10⁶ cpm/mL). Prehybridizations (1 hour at 42° C.) andhybridizations (16 hours at 42° C.) were carried out in 50% (v/v)formamide, 1 M NaCl, 1% (w/v) SDS, 10% (w/v) dextran sulphate. Filterswere washed in 2×SSC, 1% (w/v) SDS at 65° C. for 1 to 2 hours and then0.2×SSC, 1% (w/v) SDS at 65° C. for 0.5 to 1 hour. Filters were exposedto Kodak XAR film with an intensifying screen at −70° C. for 16 hours.

RFLP and Northern Analysis of the Cytochrome P450 Fragments

RFLP analysis was used to investigate linkage of the genes correspondingto the OGR-27, OGR-38 and OGR-39 cDNA clones to the Ht1 locus.

³²P-labelled fragments of OGR-27, OGR-38 and OGR-39 cDNA clones wereused to probe RNA blots and Southern blots of genomic DNA isolated fromindividual plants in the VR×V23 backcross population. Analysis of EcoRIdigested genomic DNA isolated from a VR×V23 backcross populationrevealed a RFLP for the OGR-38 probe which was linked to Ht1.Furthermore, a much reduced level of transcript was detected in theV23-like lines, when compared with the high levels of transcriptdetected in VR-like lines (FIG. 6).

The data provided strong evidence that the OGR-38 cDNA clone, containedin plasmid pCGP1805, corresponded to the Ht1 locus and represented aF3′H.

RFLP Analysis of a V23×R51 F₂ Backcross

RFLP analysis was used to investigate linkage of the gene correspondingto the OGR-38 cDNA to known genetic loci.

The RFLP linkage analysis was performed using a Macintosh version 2.0 ofthe MapMaker mapping program (Du Pont) (Lander et al, 1987). A LOD scoreof 3.0 was used for the linkage threshold.

Analysis of EcoRI or XbaI digested genomic DNA isolated from a V23×R51F₂ population revealed a RFLP for the OGR-38 probe which was linked toPAc4. PAc4, a petunia actin cDNA clone (Baird and Meagher, 1987), is amolecular marker for chromosome III and is linked to the HtI locus(McLean et al., 1990). There was co-segregation of the OGR-38 and PAc4RFLPs for 36 out of 44 V23×R51 F₂ plants. This represents arecombination frequency of 8% which is similar to a reportedrecombination frequency of 16% between the Ht1 locus and PAc4 (Cornu etal., 1990).

Further Characterisation of OGR-38

The developmental expression profiles in OGR petals, as well as in otherOGR tissues, were determined by using the ³²P-labelled fragments of theOGR-38 cDNA insert as a probe against an RNA blot containing 20 μg oftotal RNA isolated from each of the five petunia OGR petal developmentalstages as well as from leaves, sepals, roots, stems, peduncles, ovaries,anthers and styles. The OGR-38 probe hybridized with a 1.8 kb transcriptthat peaked at the younger stages of 1 to 3 of flower development. TheOGR-38 hybridizing transcript was most abundant in the petals andovaries and was also detected in the sepals, peduncles and anthers ofthe OGR plant. A low level of transcript was also detected in the stems.Under the conditions used, no hybridizing transcript was detected byNorthern analysis of total RNA isolated from leaf, style or roots.

EXAMPLE 6 Complete Sequence of OGR-38

The complete sequence of the OGR-38 cDNA clone (SEQ ID NO: 1) wasdetermined by compilation of sequence from different pUC18 subclonesobtained using standard procedures for the generation ofrandomly-overlapping clones (Sambrook et al., 1989). The sequencecontained an open reading frame of 1536 bases which encodes a putativepolypeptide of 512 amino acids.

The nucleotide and predicted amino acid sequences of OGR-38 (SEQ ID NO:1 and SEQ ID NO:2) were compared with those of the cytochrome P450probes used in the screening process and with other petunia cytochromeP450 sequences (U.S. Pat. No. 5,349,125) using an 1fasta alignment(Pearson and Lipman, 1988). The nucleotide sequence of OGR-38 was mostsimilar to the nucleic acid sequence of the flavonoid 3′ 5′-hydroxylasesrepresenting Hf1 and Hf2 loci from P. hybrida (Holton et al., 1993). TheHf1 clone showed 59.6% similarity to the OGR-38 cDNA clone, over 1471nucleotides, and 49.9% similarity, over 513 amino acids, while the Hf2clone showed 59.1% similarity to the OGR-38 cDNA clone, over 1481nucleotides, and 49.0% similarity, over 511 amino acids.

EXAMPLE 7 The F3′H Assay of the Ht1 cDNA Clone (OGR-38) Expressed inYeast Construction of pCGP1646

The plasmid pCGP1646 (FIG. 7) was constructed by cloning the OGR-38 cDNAinsert from pCGP1805 in a “sense” orientation behind the yeastglyceraldehyde-3-phosphate dehydrogenase promoter of pYE22m (Tanaka etal., 1988).

The plasmid pCGP1805 was linearised by digestion with Asp718. Theoverhanging 5′ ends were “filled in” using DNA polymerase (Klenowfragment) according to standard protocols (Sambrook et al., 1989). The1.8 kb OGR-38 cDNA fragment was released upon digestion with SmaI. ThecDNA fragment was isolated and purified using the Bresaclean kit(Bresatec) and ligated with blunted EcoRI ends of pYE22m. The plasmidpYE22m had been digested with EcoRI and the overhanging 5′ ends wereremoved using DNA polymerase (Klenow fragment) according to standardprotocols (Sambrook et al., 1989). The ligation was carried with theAmersham Ligation kit using 100 ng of the 1.8 kb OGR-38 fragment and 150ng of the prepared yeast vector, pYE22m. Correct insertion of the insertin pYE22m was established by XhoI/SalI restriction enzyme analysis ofthe plasmid DNA isolated from ampicillin-resistant transformants.

Yeast Transformation

The yeast strain G-1315 (Mat α, trpl) (Ashikari et al., 1989) wastransformed with pCGP1646 according to Ito et al. (1983). Thetransformants were selected by their ability to restore G-1315 totryptophan prototrophy.

Preparation of Yeast Extracts for Assay of F3′H Activity

A single isolate of G-1315/pCGP1646 was used to inoculate 50 mL ofModified Burkholder's medium (20.0 g/L dextrose, 2.0 g/L L-asparagine,1.5 g/L KH₂PO₄, 0.5 g/L MgSO₄.7H₂O, 0.33 g/L CaCl₂, 2 g/L (NH₄)₂SO₄, 0.1mg/L KI, 0.92 g/L (NH4)₆Mo7O₂4.4H₂O, 0.1 g/L nitrilotriacetic acid, 0.99mg/L FeSO₄.7H₂O, 1.25 mg/L EDTA, 5.47 mg/L ZnSO₄.7H₂O, 2.5 mg/LFeSO₄.7H₂O, 0.77 mg/L MnSO₄.7H₂O, 0.196 mg/L CuSO₄.5H₂O, 0.124 mg/LCo(NH₄)₂(SO₄)₂.6H₂O, 0.088 mg/L Na₂B₄O₇. 10H₂O, 0.2 mg/L thiamine, 0.2mg/L pyridoxine, 0.2 mg/L nicotinic acid, 0.2 mg/L pantothenate, 0.002mg/L biotin, 10 mg/L-inositol) which was subsequently incubated untilthe value at OD₆₀₀ was 1.8 at 30° C. Cells were collected bycentrifugation and resuspended in Buffer 1 [10 mM Tris-HCl buffer (pH7.5) containing 2 M sorbitol, 0.1 mM DTT, 0.1 mM EDTA, 0.4 mMphenylmethylsulfonyl fluoride (PMSF) and 5 mg yeast lytic enzyme/mL].Following incubation for 1 hour at 30° C. with gentle shaking, the cellswere pelleted by centrifugation and washed in ice cold Buffer 2 [10 mMTris-HCl (pH7.5) containing 0.65 M sorbitol, 0.1 mM DTT, 0.1 mM EDTA,0.4 mM PMSF]. The cells were then resuspended in Buffer 2 and sonicatedusing six 15-second bursts with a Branson Sonifier 250 at duty cycle 30%and output control 10%. The sonicated suspension was centrifuged at10,000 rpm for 30 minutes and the supernatant was centrifuged at 13,000rpm for 90 minutes. The microsomal pellet was resuspended in assaybuffer (100 mM potassium phosphate (pH 8), 1 mM EDTA, 20 mM2-mercaptoethanol) and 100 μL was assayed for activity.

F3′H Assay

F3′H enzyme activity was measured using a modified version of the methoddescribed by Stotz and Forkmann (1982). The assay reaction mixturetypically contained 100 μL of yeast extract, 5 μL of 50 mM NADPH inassay buffer (100 mM potassium phosphate (pH8.0), 1 mM EDTA and 20 mM2-mercaptoethanol) and 10 μCi of [³H]-naringenin and was made up to afinal volume of 210 μL with the assay buffer. Following incubation at23° C. for 2-16 hours, the reaction mixture was extracted with 0.5 mL ofethylacetate. The ethylacetate phase was dried under vacuum and thenresuspended in 10 μL of ethylacetate. The tritiated flavonoid moleculeswere separated on cellulose thin layer plates (Merck Art 5577, Germany)using a chloroform: acetic acid: water (10:9:1 v/v) solvent system. Thereaction products were localised by autoradiography and identified bycomparison to non-radioactive naringenin and eriodictyol standards whichwere run alongside the reaction products and visualised under UV light.

F3′H activity was detected in extracts of G1315/pCGP1646, but not inextracts of non-transgenic yeast. From this it was concluded that thecDNA insert from pCGP1805 (OGR-38), which was linked to the Ht1 locus,encoded a F3′H.

EXAMPLE 8 Transient Expression of the Ht1 cDNA Clone (OGR-38) in PlantsConstruction of pCGP1867

Plasmid pCGP1867 (FIG. 8) was constructed by cloning the cDNA insertfrom pCGP1805 in a “sense” orientation behind the Mac promoter (Comai etal., 1990) of pCGP293 (Brugliera et al., 1994). The plasmid pCGP1805 wasdigested with XbaI and KpnI to release the cDNA insert. The cDNAfragment was isolated and purified using the Bresaclean kit (Bresatec)and ligated with XbaI/KpnI ends of the pCGP293 binary vector. Theligation was carried out using the Amersham ligation kit. Correctinsertion of the fragment in pCGP1867 was established by XbaI/KpnIrestriction enzyme analysis of DNA isolated from gentamycin-resistanttransformants.

Transient Expression of the Ht1 cDNA Clone (OGR-38) in Petunia Petals

In order to rapidly determine whether the OGR-38 cDNA fragment inpCGP1867 represented a functional F3′H in plants, a transient expressionstudy was established. Petals of the mutant P. hybrida line Skr4×SW63were bombarded with gold particles (1 μm diameter) coated with pCGP1867DNA.

Gold microcarriers were prewashed 3 times in 100% ethanol andresuspended in sterile water. For each shot, 1 μg of pCGP1867 DNA, 0.5mg of gold microcarriers, 10 μL of 2.5 M CaCl₂ and 2 μL of 100 mMspermidine (free base) were mixed by vortexing for 2 minutes. The DNAcoated gold particles were pelleted by centrifugation, washed twice with100% ethanol and finally resuspended in 10 μL of 100% ethanol. Thesuspension was placed directly on the centre of the macrocarrier andallowed to dry.

Stages 1 and 2 of Skr4×SW63 flowers were cut vertically into halves andpartially embedded in MS solid media (3% (w/v) sucrose, 100 mg/Lmyo-inositol, 1×MS salts, 0.5 mg/L pyridoxine-HCl, 0.1 mg/Lthiamine-HCl, 0.5 mg/L nicotinic acid and 2 mg/L glycine). The petalswere placed so that the inside of the flower buds were facing upwards. ABiolistic PDS-1000/He System (Bio-Rad), using a Helium gas pressure of900 psi and a chamber vacuum of 28 inches of mercury, was used toproject the gold microcarriers into the petal tissue. After 6-12 hoursunder lights in a controlled plant growth room at 22° C., redanthocyanin spots were observed on the upper epidermal layer of thepetal tissue bombarded with pCGP1867-coated particles. No coloured spotswere observed in control petal bombarded with gold particles alone.These results indicated that the OGR-38 cDNA clone under the control ofthe Mac promoter was functional, at least transiently, in petal tissue.

EXAMPLE 9 Stable Expression of the Ht1 cDNA Clone (OGR-38) in PetuniaPetals—Complementation of a ht1/ht1 Petunia Cultivar

A. tumefaciens Transformations

The plasmid pCGP1867 (FIG. 8) was introduced into the Agrobacteriumtumefaciens strain AGL0 by adding 5 μg of plasmid DNA to 100 μL ofcompetent AGL0 cells prepared by inoculating a 50 mL MG/L (Garfinkel andNester, 1980) culture and growing for 16 hours with shaking at 28° C.The cells were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100mM CaCl₂/15% (v/v) glycerol. The DNA-Agrobacterium mixture was frozen byincubation in liquid N2 for 2 minutes and then allowed to thaw byincubation at 37° C. for 5 minutes. The DNA/bacterial mix was thenplaced on ice for a further 10 minutes. The cells were then mixed with 1mL of LB (Sambrook et al., 1989) media and incubated with shaking for 16hours at 28° C. Cells of A. tumefaciens carrying pCGP1867 were selectedon LB agar plates containing 10 μg/mL gentamycin. The presence ofpCGP1867 was confirmed by Southern analysis of DNA isolated from thegentamycin-resistant transformants.

Petunia Transformations

(a) Plant Material

Leaf tissue from mature plants of P. hybrida cv Skr4×SW63 was treated in1.25% (w/v) sodium hypochlorite for 2 minutes and then rinsed threetimes in sterile water. The leaf tissue was then cut into 25 mm² squaresand precultured on MS media (Murashige and Skoog, 1962) supplementedwith 0.05 mg/L kinetin and 1.0 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D) for 24 hours.

(b) Co-cultivation of Agrobacterium and Petunia Tissue

A. tumefaciens strain AGL0 containing the binary vector pCGP1867 (FIG.11) was maintained at 4° C. on MG/L agar plates with 100 mg/Lgentamycin. A single colony was grown overnight in liquid mediumcontaining 1% (w/v) Bacto-peptone, 0.5% (w/v) Bacto-yeast extract and 1%(w/v) NaCl. A final concentration of 5×10⁸ cells/mL was prepared thenext day by dilution in liquid MS medium containing B5 vitamins (Gamborget al., 1968) and 3% (w/v) sucrose (BPM). The leaf discs were dipped for2 minutes into BPM containing AGL0/pCGP1867. The leaf discs were thenblotted dry and placed on co-cultivation media for 4 days. Theco-cultivation medium consisted of SH medium (Schenk and Hildebrandt,1972) supplemented with 0.05 mg/L kinetin and 1.0 mg/L 2,4-D andincluded a feeder layer of tobacco cell suspension spread over theco-cultivation medium with a filter paper placed on top of the tobaccocell suspension.

(c) Recovery of Transgenic Petunia Plants

After co-cultivation, the leaf discs were transferred to selectionmedium (MS medium supplemented with 3% (w/v) sucrose,α-benzylaminopurine (BAP) 2 mg/L, 0.5 mg/L α-naphthalene acetic acid(NAA), kanamycin 300 mg/L, 350 mg/L cefotaxime and 0.3% (w/v) GelriteGellan Gum (Schweizerhall)). Regenerating explants were transferred tofresh selection medium after 4 weeks. Adventitious shoots which survivedthe kanamycin selection were isolated and transferred to BPM containing100 mg/L kanamycin and 200 mg/L cefotaxime for root induction. Allcultures were maintained under a 16 hour photoperiod (60 μmol. m-2, s-1cool white fluorescent light) at 23±2° C. When roots reached 2-3 cm inlength the transgenic petunia plantlets were transferred to autoclavedDebco 51410/2 potting mix in 8 cm tubes. After 4 weeks, plants werereplanted into 15 cm pots, using the same potting mix, and maintained at23° C. under a 14 hour photoperiod (300 μmol. m-2, s-1 mercury halidelight).

EXAMPLE 10 Transgenic Plant Phenotype Analysis

pCGP1867 in Skr4×SW63

Table 5 shows the various petal and pollen colour phenotypes obtainedwith Skr4×SW63 plants transformed with the pCGP1867 plasmid. Thetransgenic plants #593A, 590A, 571A, 589A, 592A and 591A producedflowers with altered petal colour. Moreover, the anthers and pollen ofthe flowers from plants #593A, 590A, 589A, 592A and 591A were pink,compared with those of the control Skr4×SW63 plant, which were white.The change in anther and pollen colour, observed on introduction ofplasmid pCGP1867 into Skr4×SW63 petunia plants, was an unanticipatedoutcome. The colour codes are taken from the Royal HorticulturalSociety's Colour Chart (RHSCC). They provide an alternative means bywhich to describe the colour phenotypes observed. The designatednumbers, however, should be taken only as a guide to the perceivedcolours and should not be regarded as limiting the possible colourswhich may be obtained. TABLE 5 Summary of petal, anther and pollencolours obtained in Skr4 × SW63 plants transformed with pCGP1867 Anther& Accession RHSCC Code Pollen Number Petal Limb Colour (petal limb)Colour Skr4 × very pale lilac 69B/73D white SW63 control (594A) 593Adark pink 67B pink 590A dark pink and pink sectors sectored 67B and pink73A 571A pink 68A and B pink 589A dark pink 68A pink 592A pink and lightpink sectors 68A and 68B light pink 591A dark pink 68A pink 570A verypale lilac 69B/73D white

The expression of the introduced Ht1 cDNA in the Skr4×SW63 hybrid had amarked effect on flower colour. The stamen tissue of the non-transgeniccontrol is white, whereas the same tissue in most of the transgenicplants was pink. In addition, expression of the Ht1 cDNA in theSkr4×SW63 hybrid conferred a dark pink hue to the corolla, which isnormally very pale lilac.

EXAMPLE 11 Analysis of Products

The anthocyanidins and flavonols produced in the petals and stamens(included the pollen, anthers and filaments) of the Skr4×SW63 plantstransformed with pCGP1867 were analysed by TLC.

Extraction of Anthocyanins and Flavonols

Prior to TLC analysis, the anthocyanin and flavonol molecules present inpetal and stamen extracts were acid hydrolysed to remove glycosylmoieties from the anthocyanidin or flavonol core. Anthocyanidin andflavonol standards were used to help identify the compounds present inthe floral extracts.

Anthocyanins and flavonols were extracted and hydrolysed by boilingbetween 100 to 200 mg of petal limbs, or five stamens, in 1 mL of 2 Mhydrochloric acid for 30 minutes. The hydrolysed anthocyanins andflavonols were extracted with 200 μL of iso-amylalcohol. This mixturewas then dried down under vacuum and resuspended in a smaller volume ofmethanol/1% (v/v) HCl. The volume of methanol/1% (v/v) HCl used wasbased on the initial fresh weight of the petal so that the relativelevels of flavonoids in the petals could be estimated. Extracts from thestamens were resuspended in 1 μL of methanol/1% (v/v) HCl. A 1 μLaliquot of the extracts from the pCGP1867 in Skr4×SW63 petals andstamens was spotted onto a TLC plate.

TLC Analysis of Floral Extracts

Acid-hydrolysed floral extracts were run in a Forestal solvent system(HOAc: water: HCl; 30: 10: 3) (Markham, 1982). Table 6 shows the resultsof the TLC analysis of the anthocyanidins and flavonols present in someof the flowers and stamens of the transgenic Skr4×SW63 petunia plantstransformed with pCGP1867. Indicative relative amounts of the flavonolsand anthocyanidins (designated with a “+” to “+++”) were estimated bycomparing the intensities of the spots observed on the TLC plate. TABLE6 Relative levels of anthocyanidins and flavonols detected in the petallimbs and stamens of Skr4 × SW63 plants transformed with pCGP1867. PetalAnthocyanidins Flavonols Acc# Colour Malvidin Cyanidin PeonidinKaempferol Quercetin Skr4 × SW63 pale lilac +/− − − + − control petallimb 593A petal limb dark pink − + +++ − ++ 571A petal limb pink − + +− + 589A petal limb dark pink − + ++ − ++ 570A petal limb pale lilac +/−− − + − Skr4 × SW63 white − − − +++ + control stamens 593A stamens pink− − ++ − +++

Introduction of the Ht1 cDNA clone into Skr4×SW63 led to production ofthe 3′-hydroxylated flavonoids, quercetin, peonidin and some cyanidin inthe petals. Peonidin is the methylated derivative of cyanidin (FIGS. 1 aand 1 b). Only kaempferol and a small amount of malvidin were detectedin the non-transgenic Skr4×SW63 control (Table 6). Although Skr4×SW63 ishomozygous recessive for both the Hf1 and Hf2 genes, these mutations donot completely block production of F3′5′ H (see U.S. Pat. No. 5,349,125)and low levels of malvidin are produced to give the petal limb a palelilac colour.

The stamens with the pink pollen and anthers produced by the transgenicplant #593A contained peonidin and quercetin, while the non-transgenicSkr4×SW63 control with white pollen and anthers contained kaempferol anda low level of quercetin (Table 6).

The accumulation of the 3′-hydroxylated anthocyanidin, peonidin, in thepetals and stamens of the transgenic Skr4×SW63/pCGP1867 plantscorrelated with the pink and dark pink colours observed in the petals,anthers and pollen of the same plants.

Co-Suppression of F3′H Activity

The plasmid pCGP1867 was also introduced into P. hybrida cv. Old GloryRed (Ht1) in order to reduce the level of F3′H activity.

Petunia transformations were carried out as described in Example 9,above.

Two out of 38 trangenic plants produced flowers with an alteredphenotype. OGR normally produces deep red flowers (RHSCC#46B). The twotransgenic plants with altered floral colour produced flowers with alight pink or light red hue (RHSCC#54B and #53C).

Northern analysis on RNA isolated from flowers produced by fourtransgenic plants (the two transgenics with an altered phenotype and twotransgenics with the usual deep red flowers) was performed to examinethe level of OGR-38 transcripts. Ten micrograms of total petal RNA wasseparated on a 1.2% (w/v) agarose/formaldehyde gel (Sambrook et al.1989) and transferred to HybondN nylon membrane (Amersham), as describedpreviously. Petal RNA from a non-transformed OGR flower was alsoincluded as a control. ³²P-labelled fragments of the OGR-38 cDNA insertswere used to probe the RNA blot.

The OGR-38 probe detected transcripts of approximately 2.4 kb and 1.8 kbin the flowers of the transgenic plants. However, the level of bothtranscripts detected in the light pink and light red flowers wasconsiderably lower than that detected in the deep red transgenicflowers. The endogenous 1.8 kb transcript was also detected in RNA fromthe non-transformed OGR flowers. In order to confirm that the 2.4 kbtranscript was from the introduced OGR-38 transgene, ³²P-labelledfragments of the mas terminator region were used to probe the same RNAblot. The mas probe detected the 2.4 kb transcript, suggesting that atleast this transcript was derived from the introduced OGR-38 transgene.

Analysis of Anthocyanin Levels

The levels of anthocyanins in the control flowers and in the light pinktransgenic flower were measured by spectrophotometric analysis.

Extraction of Anthocyains and Flavonols

Anthocyanins and flavonols were extracted from petal limbs by incubating200 to 300 mg of petal limb in 2 mL of methanol/1% (v/v) HCl for 16hours at 4° C. Fifty μL of this solution was then added to 950 μL ofmethanol/1% (v/v) HCl and the absorbance of the diluted solution at 530nm was determined. The anthocyanin level in nmoles per gram wasdetermined using the formula: [(Abs (530 nm)/34,000)×volume ofextraction buffer×dilution factor×10⁶]/weight in grams.

The light pink flower was found to contain approximately 915 nmoles ofanthocyanin per gram of petal limb tissue whilst the control flowercontained around 4000 nmoles/gram.

These data suggest that introduction of the petunia F3′H (OGR-38) cDNAclone in a sense orientation into OGR plants leads to “co-suppression”(i.e. reduction) of both the endogenous and the transgenic F3′Htranscripts. A correlation was observed between lighter flower colours,reduced anthocyanin production and reduced F3′H transcript level.

EXAMPLE 12 Isolation of a F3′H cDNA Clone From Dianthus Caryophyllus

In order to isolate a Dianthus caryophyllus (carnation) F3′H cDNA clone,the petunia Ht1-linked F3′H cDNA clone (OGR-38), contained in pCGP1805(described above), was used to screen a Carnation cv. Kortina Chanelpetal cDNA library, under low stringency conditions.

Construction of Carnation cv. Kortina Chanel cDNA Library

Twenty micrograms of total RNA isolated (as described previously) fromstages 1, 2 and 3 of Kortina Chanel flowers was reverse transcribed in a50 μL volume containing 1×Superscript™ reaction buffer, 10 mMdithiothreitol (DTT), 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 500 μM5-methyl-dCTP, 2.8 μg Primer-Linker oligo from ZAP-cDNA Gigapack IIIGold cloning kit (Stratagene) and 2 μL Superscript™ reversetranscriptase (BRL). The reaction mix was incubated at 37° C. for 60minutes, then placed on ice. A ZAP-cDNA Gigapack III Gold Cloning kit(Stratagene) was used to complete the library construction. The totalnumber of recombinants was 2.4×10⁶.

A total of 200,000 pfu of the packaged cDNA was plated at 10,000 pfu per15 cm diameter plate after transfecting XL1-Blue MRF′ cells. The plateswere incubated at 37° C. for 8 hours, then stored overnight at 4° C.Duplicate lifts were taken onto Colony/Plaque Screen™ filters (DuPont)and treated as recommended by the manufacturer.

Screening of Kortina Chanel Petal cDNA Library for a F3′H cDNA Clone

Prior to hybridization, the duplicate plaque lifts were treated asdescribed previously. The duplicate lifts from the Kortina Chanel petalcDNA library were screened with ³²P-labelled fragments of the 1.8 kbEcoRI/XhoI insert from pCGP1805. Low stringency conditions, as describedfor the screening of the petunia OGR cDNA library, were used.

One strongly-hybridizing plaque was picked into PSB and rescreened asdetailed above to isolate purified plaques. The plasmid contained in the1ZAP bacteriophage vector was rescued and named pCGP1807.

The KC-1 cDNA insert contained in pCGP1807 was released upon digestionwith EcoRI/XhoI and is around 2 kb. The complete sequence of the KC-1cDNA clone was determined by compilation of sequence from subclones ofthe KC-1 cDNA insert. (Partial sequence covering 458 nucleotides hadpreviously been generated from a 800 bp KpnI fragment covering the 3′region of KC-1 which was subcloned into pBluescript to give pCGP1808.)The complete sequence (SEQ ID NO:3) contained an open reading frame of1508 bases which encodes a putative polypeptide of 500 amino acids (SEQID NO:4).

The nucleotide and predicted amino acid sequences of the carnation KC-1cDNA clone were compared with those of the petunia OGR-38 F3′H cDNAclone (SEQ ID NO: 1 and SEQ ID NO:2). The sequences of the carnationKC-1 cDNA clone (SEQ ID NO:3 and 4) showed 67.3% similarity, over 1555nucleotides, and 71.5% similarity, over 488 amino acids, to that of thepetunia OGR-38 F3′H cDNA clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 13 Stable Expression of the Carnation F3′H cDNA (KC-1) Clone inPetunia Petals—Complementation of a ht1/ht1 Petunia Cultivar

Preparation of pCGP1810

Plasmid pCGP1810 (FIG. 9) was constructed by cloning the cDNA insertfrom pCGP1807 in a “sense” orientation behind the Mac promoter (Comai etal., 1990) of pCGP90 (U.S. Pat. No. 5,349,125), a pCGP293 basedconstruct (Brugliera et al., 1994). The plasmid pCGP1807 was digestedwith BamHI and ApaI to release the KC-1 cDNA insert. The cDNA fragmentwas isolated and purified using the Bresaclean kit (Bresatec). ThepCGP90 binary vector was digested with BamHI and ApaI to release thelinearised vector and the Hf1 cDNA insert. The linearised vector wasisolated and purified using the Bresaclean kit (Bresatec) and ligatedwith BamHI/ApaI ends of the KC-1 cDNA clone. The ligation was carriedout using the Amersham ligation. Correct insertion of the insert inpCGP1810 was established by BamHI/ApaI restriction enzyme analysis ofDNA isolated from gentamycin-resistant transformants.

The binary vector pCGP1810 was introduced into A. tumefaciens strainAGL0 cells, as described in Example 9. The pCGP1810/AGL0 cells weresubsequently used to transform. Skr4×SW63 petunia plants (also describedin Example 9), to test for stable expression and activity of the enzymeencoded by the gene corresponding to the KC-1 cDNA clone.

EXAMPLE 14 Transgenic Plant Phenotype Analysis

pCGP1810 in Skr4×SW63

The expression of the introduced KC-1 cDNA in the Skr4×SW63 hybrid had amarked effect on flower colour. Ten of the twelve transgenic plantstransformed with pCGP1810 produced flowers with an altered petal colour(RHSCC# 73A), compared with the Skr4×Sw63 control (RHSCC# 74C). Moreoverthe anthers and pollen of the transgenic flowers were pink, comparedwith those of the control Skr4×SW63 plant, which were white. Inaddition, expression of the KC-1 cDNA in the Skr4×SW63 hybrid conferreda dark pink hue to the corolla, which is normally pale lilac. The colourcodes are taken from the Royal Horticultural Society's Colour Chart(RHSCC). They provide an alternative means by which to describe thecolour phenotypes observed. The designated numbers, however, should betaken only as a guide to the perceived colours and should not beregarded as limiting the possible colours which may be obtained.

Acid-hydrolysed floral extracts (see Example 11) were run in a Forestalsolvent system (HOAc:water:HCl; 30: 10: 3) (Markham, 1982). The 3′hydroxylated flavonoids, peonidin and quercetin, were readily detectedin the petal limbs of the transgenic plants. Only kaempferol and a smallamount of malvidin were detected in the non-transgenic Skr4×SW63control.

The accumulation of the 3′-hydroxylated anthocyanidin, peonidin, in thepetals of the transgenic Skr4×SW63/pCGP1810 plants correlated with thedark pink colours observed in the petals of the same plants.

Construction of pCGP1813

Plasmid pCGP1811 was constructed by cloning the cDNA insert frompCGP1807 in a “sense” orientation behind the Mac promoter (Comai et al.,1990) of pCGP1958. The plasmid pCGP1958 contains the Mac promoter andmannopine synthase (mas)(Comai et al., 1990) terminator in a pUC19backbone. The plasmid pCGP1807 was digested with PstI and XhoI torelease the cDNA insert. The overhanging 5′ ends were filled in usingDNA polymerase (Klenow fragment) (Sambrook et al., 1989). The cDNAfragment was isolated and purified using the Bresaclean kit (Bresatec)and ligated with SmaI ends of the pCGP1958 vector to produce pCGP1811.

The plasmid pCGP1811 was subsequently digested with PstI to release theexpression cassette containing the Mac promoter driving the KC-1 cDNAwith a mas terminator, all of which were contained on a 4 kb fragment.The expression cassette was isolated and ligated with PstI ends of thepWTT2132 binary vector (DNA Plant Technology Corporation; Oakland,Calif.) to produce pCGP1813 (FIG. 10).

Transformation of Dianthus Caryophyllus cv. Kortina Chanel With theCarnation F3′H cDNA Clone.

The binary vector pCGP1813 was introduced into A. tumefaciens strainAGL0 cells, as described in Example 9. The pCGP1813/AGL0 cells were usedto transform carnation plants, to reduce the amount of 3′-hydroxylatedflavonoids.

(a) Plant Material

Dianthus caryophylls (cv. Kortina Chanel) cuttings were obtained fromVan Wyk and Son Flower Supply, Victoria, Australia. The outer leaveswere removed and the cuttings were sterilised briefly in 70% v/v ethanolfollowed by 1.25% w/v sodium hypochlorite (with Tween 20) for 6 min andrinsed three times with sterile water. All the visible leaves andaxillary buds were removed under the dissecting microscope beforeco-cultivation.

(b) Co-Cultivation of Agrobacterium and Dianthus Tissue

Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991), containingthe binary vector pCGP1813, was maintained at 4° C. on LB agar plateswith 50 mg/L tetracycline. A single colony was grown overnight in liquidLB broth containing 50 mg/L tetracycline and diluted to 5×10⁸ cells/mLnext day before inoculation. Dianthus stem tissue was co-cultivated withAgrobacterium for 5 days on MS medium supplemented with 3% w/v sucrose,0.5 mg/L BAP, 0.5 mg/L 2,4-dichlorophenoxy-acetic acid (2,4-D), 100 mMacetosyringone and 0.25% w/v Gelrite (pH 5.7).

(c) Recovery of Transgenic Dianthus Plants

For selection of transformed stem tissue, the top 6-8 mm of eachco-cultivated stem was cut into 3-4 mm segments, which were thentransferred to MS medium (Murashige and Skoog, 1962) supplemented with0.3% w/v sucrose, 0.5 mg/L BAP, 0.5 mg/L 2,4-D, 1 μg/L chlorsulfuron,500 mg/L ticarcillin and 0.25% w/v Gelrite. After 2 weeks, explants weretransferred to fresh MS medium containing 3% sucrose, 0.16 mg/Lthidiazuron (TDZ), 0.5 mg/L indole-3-butyric acid (IBA), 2 μg/Lchlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite and care wastaken at this stage to remove axillary shoots from stem explants. After3 weeks, healthy adventitious shoots were transferred to hormone free MSmedium containing 3% w/v sucrose, 5 μg/L chlorsulfuron, 500 mg/Lticarcillin, 0.25% w/v Gelrite. Shoots which survived 5 μg/Lchlorsulfuron were transferred to the same medium for shoot elongation.

Elongated shoots were transferred to hormone-free MS medium containing 5μg/L chlorsulfuron, 500 mg/L ticarcillin and 0.4% w/v Gelrite, in glassjars, for normalisation and root production. All cultures weremaintained under a 16 hour photoperiod (120 mE/m²/s cool whitefluorescent light) at 23±2° C. Normalised plantlets, approximately 1.5-2cm tall, were transferred to soil (75% perlite/25% peat) for acclimationat 23° C. under a 14 hour photoperiod (200 mE/m²/s mercury halide light)for 3-4 weeks. Plants were fertilised with a carnation mix containing 1g/L CaNO₃ and 0.75 g/L of a mixture of microelements plus N:P:K in theratio 4.7:3.5:29.2.

EXAMPLE 15 Isolation of a F3′H cDNA Clone from Antirrhinum Majus(Snapdragon) Using a Differential Display Approach

A novel approach was employed to isolate a cDNA sequence encoding F3′Hfrom Antirrhinum majus (snapdragon). Modified methods based on theprotocols for (i) isolation of plant cytochrome P450 sequences usingredundant oligonucleotides (Holton et al. 1993) and (ii) differentialdisplay of eukaryotic messenger RNA (Liang and Pardee, 1992) werecombined, to compare the petal cytochrome P450 transcript populationsbetween wild type (Eos⁺) and F3′H mutant (eos⁻)snapdragon lines. Directcloning of differentially expressed cDNA fragments allowed their furthercharacterisation by Northern, RFLP and sequence analysis to identifyputative F3′H encoding sequences. A full-length cDNA was obtained usingthe RACE protocol of Frohman et al. (1988) and the clone was shown toencode a functional F3′H following both transient and stable expressionin petunia petal cells.

Plant Material

The Antirrhinum majus lines used were derived from the parental linesK16 (eos⁻) and N8 Eos⁺). K16 is a homozygous recessive mutant lackingF3′H activity, while N8 is wild type for F3′H activity. These lines aresimilar, though not isogenic. The seed of capsule E228² from the selfedK16×N8 F₁ plant (#E228) was germinated and the resultant plants (K16×N8F₂ plants) were scored for the presence or absence of cyanidin, aproduct of F3′H activity (see FIGS. 1 a and 1 b). The presence ofcyanidin could be scored visually, as the flowers were a crimson colour,unlike the mutant plants which were a pink colour (frompelargonidin-derived pigments). The accuracy of the visual scoring wasconfirmed by TLC analysis of the petal anthocyanins, carried out asdescribed in Example 11.

Of 13 plants raised from the E228² seed, 9 (#3, #4, #5, #6, #7, #9, #10,#12, #15) produced flowers with cyanidin (Eos⁺/Eos⁺ and Eos⁺/eos⁻) while4 (#8, #11, #13, #14) produced only pelargonidin-derived pigments(eos⁻/eos⁻).

Synthesis of cDNA

Total RNA was isolated from the leaves of plant #13 and petal tissue ofplants #3, #5, and #12 of the A. majus K16×N8 F₂ segregating population(E228²) using the method of Turpen and Griffith (1986). ContaminatingDNA was removed by treating 50 μg total RNA with 1 unit RQ1 RNase-freeDNase (Promega) in the presence of 40 units RNasin® ribonucleaseinhibitor (Promega) for 3 hours at 37° C. in a buffer recommended by themanufacturers. The RNA was then further purified by extraction withphenol/chloroform/iso-amyl alcohol (25:24:1) and subsequent ethanolprecipitation.

Anchored poly(T) oligonucleotides, complementary to the upstream regionof the polyadenylation sequence, were used to prime cDNA synthesis fromA. majus petal and leaf RNA. The oligonucleotide sequences synthesizedwere (5′-3′): polyT-anchA TTTTTTTTTTTTTTTTTA SEQ ID NO: 27 polyT-anchCTTTTTTTTTTTTTTTTTC SEQ ID NO: 28 polyT-anchG TTTTTTTTTTTTTTTTTG SEQ IDNO: 29

Two micrograms of total RNA and 100 pmol of the appropriate primingoligonucleotide were heated to 70° C. for 10 minutes, then chilled onice. The RNA/primer hybrids were then added to a reaction containing 20units RNasin® (Promega), 25 nM each dNTP, 10 mM DTT and 1× Superscriptbuffer (BRL). This reaction was heated at 37° C. for 2 minutes, then 200units of Superscript™ reverse transcriptase (BRL) were added and thereaction allowed to proceed for 75 minutes, after which the reversetranscriptase was inactivated by heating the mixture at 95° C. for 20minutes.

Amplification of Cytochrome P450 Sequences using PCR

Cytochrome P450 sequences were amplified using redundantoligonucleotides (designed to be complementary to conserved regions nearthe 3′ end of plant cytochrome P450 coding sequences) and polyT anchoredoligonucleotides. A similar approach was previously used to generatecytochrome P450 sequences from Petunia hybrida and is described in U.S.Pat. No. 5,349,125.

Four oligonucleotides (referred to as upstream primers) weresynthesized. These were derived from conserved amino acid regions inplant cytochrome P450 sequences. The oligonucleotides (written 5′ to 3′)were as follows: WAIGRDP TGG GCI ATI GGI (A/C)GI GA(T/C) CC SEQ ID NO:30 SEQ ID NO: 31 FRPERF AGG AAT T(T/C)(A/C) GIC CIG A(A/G)(A/C) GIT TSEQ ID NO: 32 SEQ ID NO: 33 Pet Haem-New CCI TT(T/C) GGI GCI GGI (A/C)GI(A/C)GI ATI TG(T/G) (C/G)CI GG SEQ ID NO: 34 EFXPERF GAI TT(T/C) III CCIGAI (A/C)GI TT SEQ ID NO: 35 SEQ ID NO: 36

The upstream primers were used with each of the polyT anchoredoligonucleotides to generate cytochrome P450 sequences in polymerasechain reactions using cDNA as a template. Fifty pmol of eacholigonucleotide was combined with 2 μM of each dNTP, 1.5 mM MgCl₂, 1×PCRbuffer (Perkin Elmer), 5 μCi α-[³³P] dATP (Bresatec, 1500 Ci/mmol), 2.5units AmpliTaq® DNA polymerase (Perkin Elmer) and {fraction (1/10)}th ofthe polyT-anchor primed cDNA reaction (from above). Reaction mixes (50μL) were cycled 40 times between 94° C. for 15 seconds, 42° C. for 15seconds, and 70° C. for 45 seconds, following an initial 2 minutedenaturation step at 94° C. Cycling reactions were performed using aPerkin Elmer 9600 Gene Amp Thermal Cycler.

DNA sequences were amplified using each upstream primer/anchored primercombination and the appropriately-primed cDNA template. Each primercombination was used with the cDNA from the petals of the E228² plants#3 and #5 (cyanidin-producing flowers) and #12 (non-cyanidin producingflowers). Reactions incorporating leaf cDNA from plant #13(cyanidin-producing flowers) were also included, as negative controls,because F3′H activity is not present at a significant level in healthy,unstressed leaf tissues.

Differential Display of Cytochrome P450 Sequences

³³P-labelled PCR fragments were visualised following separation on a 5%(w/v) polyacrylamide/urea denaturing gel (Sambrook et al. 1989). A³³P-labelled M13 mp18 sequencing ladder was included on the gel to serveas a size marker. The sequencing gel was dried onto Whatman 3MM paperand exposed to Kodak XAR film at room temperature.

Comparison of bands between cyanidin-producing petal samples and thenon-cyanidin petal sample revealed 11 bands which represent mRNAsexclusively present in the cyanidin-producing petals. Of these 11 bands,only two were also present (at a reduced intensity) in the leaf sample.

Isolation and Cloning of PCR Fragments From Sequencing Gel

PCR products were purified from the dried sequencing gel and reamplifiedby the method described by Liang et al. (1993). Amplified cDNAs werepurified, following electrophoretic separation on a 1.2% (w/v)agarose/TAE gel, using a Bresaclean kit (Bresatec). The purifiedfragments were then directly ligated into either commercially-preparedpCR-Script™vector (Stratagene) or EcoRV-linearised pBluescript®(Stratagene) which had been T-tailed using the protocol of Marchuk etal. (1990).

Sequence of F3′H PCR Products

Each of the eleven cloned differential display PCR products (withinserts not exceeding 500 bp) was sequenced on both strands and comparedto other known cytochrome P450 sequences involved in anthocyaninbiosynthesis, using the FASTA algorithm of Pearson and Lipman (1988).

Of the eleven cDNAs cloned, two (Am1Gb and Am3Ga) displayed stronghomology with the petunia OGR-38 F3′H sequence (Examples 4 to 11) andthe F3′5′ H sequences (Holton et al., 1993). Conserved sequences betweenclones Am1Gb and Am3Ga suggested that they represented overlappingfragments of the same mRNA. Clone Am3Ga extends from the sequenceencoding the haem-binding region of the molecule (as recognised by the“Pet Haem-New” oligonucleotide; SEQ ID NO:34) to the polyadenylationsequence. Clone Am1Gb extends from the cytochrome P450 sequence encodingthe conserved “WAIGRDP” amino acid motif (complementary to primer 1; SEQID NO:30 and SEQ ID NO:31) to an area in the 3′ untranslated regionwhich was spuriously recognised by the primer I (“WAIGRDP”)oligonucleotide.

EXAMPLE 16 RFLP Analysis of Cytochrome P450 cDNAs

Restriction fragment length polymorphism (RFLP) analysis was again usedto investigate linkage of the gene corresponding to cDNA clone Am3Ga tothe presence, or absence, of cyanidin-producing activity in petals. A³²P-labelled insert of Am3Ga was used to probe Southern blots of genomicDNA isolated from K16×N8 F₂ segregating plants as well as the parentalK16 and N8 lines. Analysis of EcoRV-digested genomic DNA from 13 plantsof the K16×N8 F₂ segregating population revealed hybridization only tothe sequences of N8 and the K16×N8 F₂ segregating lines which displayedfloral cyanidin production (FIG. 11). The K16×N8 F₂ plants whichproduced only pelargonidin-derived pigments in their petals (includingparental line, K 16) showed no specific hybridization (FIG. 11, lanes 2,8, 11, 13, 14). These data indicate a possible deletion of the genomicsequences corresponding to Am3Ga in the mutant K16 plant and, therefore,at least a partial deletion of the F3′H gene in this line.

EXAMPLE 17 Northern Analysis of Cytochrome P450 cDNAs

Northern analysis was used to confirm the expression profiles of theputative cytochrome P450 fragments as shown by differential display. Tenmicrograms of total petal RNA from eight of the K16×N8 F₂ segregatingpopulation was separated on a 1.2% (w/v) agarose/formaldehyde gel(Sambrook et al. 1989) and transferred to HybondN nylon membrane(Amersham). Leaf RNA from the cyanidin-producing plant #13 was alsoincluded as a negative control in the Northern analysis. ³²P-labelledfragments of the cDNA insert from clone Am3Ga was used to probe the RNAblot.

The Am3Ga probe recognised an approximately 1.8 kb transcript which wasonly detectable in the petals of cyanidin-producing plants (plants #1,#3, #4, #5, #8). No transcript was detectable in thepelargonidin-producing petals (plants #6, #11, #12) or in the leafsample from plant #13 (FIG. 12).

These data, taken with those of the RFLP analysis, provide strongevidence that Am3Ga clone represents a cytochrome P450 gene which isresponsible for F3′H activity in snapdragon. The total lack of adetectable transcript in the petals of non-cyanidin-producing linessupports the findings of the RFLP analysis, that the loss ofcyanidin-producing activity in the K16 line (and the homozygousrecessive plants of the K16×N8 F₂ segregating population) is the resultof a deletion in the F3′H structural gene.

EXAMPLE 18 Isolation of a Full-Length Snapdragon F3′H cDNA

The Rapid Amplification of cDNA Ends (RACE) protocol of Frohman et al.(1988) was employed to isolate a full-length F3′H cDNA clone usingsequence knowledge of the partial Am3Ga clone. A gene-specific primer(“SnapredRace A”-complementary to Am3Ga sequences 361 to 334) wassynthesized to allow reverse transcription from petal RNA. A 3′amplification primer (“SnapredRace C”-complementary to Am3Ga (3′UTR)sequences 283 to 259) was also synthesized to bind just upstream of“SnapredRace A”. A “poly(C)” primer was used to amplify sequences fromthe 5′ end of the cDNA molecule.

The sequences of the oligonucleotides used were (written 5′-3′): SnapredRace CCA CAC GAG TAG TTT TGG CAT TTG ACC C A SEQ ID NO: 37 Snapred RaceGTC TTG GAC ATC ACA CTT CAA TCT G C SEQ ID NO: 38 PolyC race CCG AAT TCCCCC CCC CC SEQ ID NO: 39

“Snapred Race A-primed” petal cDNA was poly(G)-tailed and a 5′ cDNAfragment amplified with primers “Snapred Race C” and “PolyC race” usingthe method of Frohman et al. (1988). Pfu DNA polymerase (0.15 unit)(Stratagene) was combined with 2.5 units AmpliTaq® DNA polymerase(Perkin Elmer) to increase the fidelity of the PCR reaction.

The resultant 1.71 kb DNA fragment (sdF3′H) was cloned directly intoEcoRV-linearised pBluescript® (Stratagene) vector which had beenT-tailed using the protocol of Marchuk et al. (1990). This plasmid wasnamed pCGP246.

EXAMPLE 19 Complete Sequence of Snapdragon F3′H

Convenient restriction sites within the sdF3′H cDNA sequence of pCGP246were exploited to generate a series of short overlapping subclones inthe plasmid vector pUC19. The sequence of each of these subclones wascompiled to provide the sequence of the entire sdF3′H RACE cDNA. ThesdF3′H cDNA sequence was coupled with that from clone Am3Ga to providethe entire sequence of a snapdragon F3′H cDNA (SEQ ID NO:5). It containsan open reading frame of 1711 bases which encodes a putative polypeptideof 512 amino acids (SEQ ID NO:6).

The nucleotide and predicted amino acid sequences of the snapdragonsdF3′H clone were compared with: those of the petunia OGR-38 cDNA clone(SEQ ID NO: 1 and SEQ ID NO:2); the petunia F3′5′ H cDNA clones Hf1 andHf2; and other petunia cytochrome P450 sequences isolated previously(U.S. Pat. No. 5,349,125). The sequence of sdF3′H was most similar tothat of the petunia F3′H cDNA clone (OGR-38) representing the Ht1 locusfrom P. hybrida, having 69% similarity at the nucleic acid level, over1573 nucleotides, and 72.2% similarity at the amino acid level, over 507amino acids.

The Hf1 clone showed 57.3% similarity, over 1563 nucleotides and 49.3%similarity, over 491 amino acids, to the snapdragon sdF3′H clone, whilethe Hf2 clone showed 57.7% similarity, over 1488 nucleotides, and 50.8%similarity, over 508 amino acids, to the snapdragon sdF3′H clone.

The snapdragon sdF3′H sequence contains two “in frame” ATG codons whichcould act to initiate translation. Initiation from the first of thesecodons (position 91 of SEQ ID NO:5) gives a protein with an additional10 N-terminal amino acids and would be favoured according to thescanning model for translation (Kozak, 1989).

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 20 Transient Expression of sdF3′H in Plants

Construction of pCGP250

Plasmid pCGP250 (FIG. 13) was created by cloning the entire sdF3′H RACEcDNA insert (from position 1 to 1711 (SEQ ID NO:5)) from pCGP246 in the“sense” orientation behind the Mac promoter (Comai et al., 1990) ofpCGP293 (Brugliera et al., 1994). The plasmid pCGF246 was digested withEcoRI to release the cDNA insert. The cDNA fragment was blunt-ended byrepairing the overhangs with the Klenow fragment of DNA polymerase I(Sambrook et al., 1989) and purified, following agarose gelelectrophoresis, using a Bresaclean kit (Bresatec). The blunt cDNAfragment was then ligated into the binary vector pCGF293, which had beenlinearised with XbaI and blunt-ended using the Klenow fragment of DNApolymerase I. The ligation was carried out using the Amersham ligationkit. Correct insertion of the insert in pCGP250 was established by BamHIand PstI restriction enzyme analysis of DNA isolated fromgentamycin-resistant transformants.

Construction of pCGP231

Plasmid pCGP231 (FIG. 14) was created by cloning the RACE cDNA insertfrom pCGP246, downstream of the first “in-frame” ATG codon (fromposition 104 to 1711 (SEQ ID NO:5), in the “sense” orientation behindthe Mac promoter (Comai et al., 1990) of pCGP293 (Brugliera et al.,1994). The plasmid pCGP246 was digested with SspI (which recognises asite between the candidate ATG codons) and SmaI (with a site in thevector polylinker sequence) to release a blunt-ended cDNA fragment whichincludes the entire coding region downstream from the second putativeinitiation codon. The cDNA fragment was then ligated into the binaryvector pCGP293, which had been linearised with XbaI and blunt-endedusing the Klenow fragment of DNA polymerase I. The ligation was carriedout using the Amersham ligation kit. Correct insertion of the insert inpCGP231 was established by BamHI and PstI restriction enzyme analysis ofDNA isolated from gentamycin-resistant transformants.

Transient Expression Studies

To determine rapidly whether the pCGP246 sequences in pCGP231 andpCGP250 encoded active flavonoid 3′-hydroxylases in plants, a transientexpression study was undertaken. Petals of the mutant P. hybrida lineSkr4×SW63 were bombarded with gold particles (1 μm diameter) coated witheither pCGP231 or pCGP250 plasmid DNA, using the method described inExample 8.

After 6-12 hours under lights in a controlled plant growth room at 22°C., red anthocyanin spots were observed on the surface of the petaltissue bombarded with pCGP231 coated particles. No coloured spots wereobserved in petals bombarded with pCGP250 or control petals bombardedwith gold particles alone. These results indicated that the pCGP246coding region (starting at the second ATG, position 121 of SEQ ID NO:5),under the control of the Mac promoter, was functional in petal tissue.

EXAMPLE 21 Stable Expression of the Snapdragon F3′H cDNA Done in PetuniaPetals-Complementation of a ht1/ht1 Petunia Cultivar

The binary vectors pCGP250 and pCGP231 were introduced into A.tumeaciens strain AGL0 cells, as described in Example 9. ThepCGP250/AGL0 and pCGP231/AGL0 cells were used to transform Skr4×SW63petunia plants (also described in Example 9), to test for stableexpression and activity of the enzyme encoded by the gene correspondingto the snapdragon sdF3′H cDNA clone.

Three of the nine transgenic plants transformed with pCGP250 producedflowers with a slightly-altered petal colour (RHSCC# 73A), compared withthe Skr4×SW63 control (RHSCC# 75C). Of the 11 transgenic plantstransformed with pCGP231, one plant produced flowers with an alteredpetal colour (RHSCC# 73B). The anthers and pollen of the transgenicflowers were also white, as in the control. The codes are taken from theRoyal Horticultural Society's Colour Chart (RHSCC). They provide analternative means by which to describe the colour phenotypes observed.The designated numbers, however, should be taken only as a guide to theperceived colours and should not be regarded as limiting the possiblecolours which may be obtained.

TLC Analysis of Floral Extracts

Acid-hydrolysed floral extracts (see Example 11) were run in a Forestalsolvent system (HOAc:water:HCl; 30:10:3) (Markham, 1982). Introductionof the sdF3′H cDNA clone into Skr4×SW63 led to the production ofincreased levels of the 3′-hydroxylated flavonoid, peonidin, in thepetals. Peonidin is the methylated derivative of cyanidin (FIGS. 1 a and1 b).

EXAMPLE 22 Isolation of a F3′H cDNA Clone From Arabidopsis ThalianaUsing a PCR Approach

In order to isolate a cDNA clone representing flavonoid 3′-hydroxylasefrom Arabidopsis thaliana, PCR fragments were generated using primersfrom the conserved regions of cytochrome P450s. One PCR product(p58092.13) was found to have high sequence similarity with the petuniaOGR-38 and snapdragon F3′H cDNA clones. The PCR fragment was then used,together with the Ht1 cDNA insert (OGR-38) from pCGP1805, to screen anA. thaliana cDNA library.

Design of Oligonucleotides

Degenerate oligonucleotides for PCR DNA amplification were designed fromthe consensus amino acid sequence of Petunia hybrida cytochrome P450partial sequences situated near the haem-binding domain. Primerdegeneracy was established by the inclusion of deoxyinosine (designatedas I below) in the third base of each codon (deoxyinosine base pairswith similar efficiency to A, T, G, and C), and the inclusion ofalternate bases where the consensus sequences were non-specific. Thus,the amino-terminal directional primer “Pet Haem” (Petunia haem-bindingdomain), containing the cysteine residue codon crucial for haem binding,and the upstream primer ‘WAIGRDP’ (See also Example 15) were designed.WAIGRDP TGG GCI ATI GGI (A/C)GI GA(T/C) CC SEQ ID NO: 30 SEQ ID NO: 31Pet Haem CCI GG(A/G) CAI ATI C(G/T)(C/T) (C/T)TI CCI GCI CC(A/G) AAI GGSEQ ID NO: 40

Generation of Cytochrome P450 Sequences Using PCR

Genomic DNA was isolated from A. thaliana ecotype Columbia, using themethod described by Dellaporta et al. (1987). Polymerase chain reactionsfor amplification of cytochrome P450 homologues typically contained100-200 ng of Columbia genomic DNA, 10 mM Tris-HCl HCI (pH8.3), 50 mMKCl, 1.5 mM MgCl₂, 0.01% (w/v) gelatin, 0.2 mM each dNTP, 312 ng“WAIGRDP” and 484 ng “Pet Haem” and 1.25 units Taq polymerase (Cetus).Reaction mixes (50 μL) were cycled 40 times between 95° C. for 50seconds, 45° C. for 50 seconds and 72° C. for 45 seconds.

The expected size of specific PCR amplification products, using the“WAIGRDP” and “Pet Haem” primers on a typical P450 gene template,without an intron, is approximately 150 base pairs. PCR fragments ofapproximately 140 to 155 base pairs were isolated and purified using theMermaid® kit (BIO 101). The PCR fragments were re-amplified to obtainenough product for cloning and then end-repaired using M DNA polymeraseand finally cloned into pCR-Script™Direct SK(+) (Stratagene). Theligated DNA was then used to transform competent DH5α cells (Inoue etal., 1990).

Sequence of PCR Products

Plasmid DNA from 15 transformants was prepared (Del Sal et al., 1989).Sequencing data generated from these PCR fragments indicated that 11 outof the 15 represented unique clones. A distinct set of cytochrome P450consensus amino acids was also found in the translated sequence encodedwithin the A. thaliana PCR inserts. The sequences of the PCR fragmentswere also compared with those of the petunia OGR-38 F3′H cDNA clone andthe snapdragon F3′H cDNA clone. The PCR fragment, p58092.13, was mostsimilar to the F3′H sequences from both petunia and snapdragon.

EXAMPLE 23 Screening of A. Thaliana cDNA Library

To isolate a cDNA clone of the p58092.13 PCR product, an A. thalianaecotype Columbia cDNA library (Newman et al., 1994; D'Alessio et al.,1992) was screened with a ³²P-labelled fragment of p58092.13 togetherwith a ³²P-labelled fragment of the petunia Ht1 cDNA insert (OGR-38),contained in pCGP1805.

A total of 600,000 pfu was plated at a density of 50,000 pfus per 15 cmdiameter plate, as described by D'Alessio et al (1992). After phagegrowth at 37° C. plates were stored at 4° C. overnight, duplicate liftswere taken onto Colony/Plaque Screen™ filters (DuPont) and treated asrecommended by the manufacturer.

Prior to hybridization, the duplicate plaque lifts were washed inprewashing solution (50 mM Tris-HCl pH7.5, 1 M NaCl, 1 mM EDTA, 0.1%(w/v) sarcosine) at 65° C. for 30 minutes; stripped in 0.4 M sodiumhydroxide at 65° C. for 30 minutes; then washed in a solution of 0.2 MTris-HCl pH 8.0, 0.1×SSC, 0.1% (w/v) SDS at 65° C. for 30 minutes andfinally rinsed in 2×SSC, 1.0% (w/v) SDS.

Hybridization conditions included a prehybridization step in 50% (v/v)formamide, 1 M NaCl, 10% (w/v) dextran sulphate, 1% (w/v) SDS at 42° C.for at least 1 hour. The ³²P labelled fragment of p58092.13 (2×10⁶cpm/mL) was then added to the hybridization solution and hybridizationwas continued at 42° C. for a further 16 hours. The filters were thenwashed in 2×SSC, 1% (w/v) SDS at 42° C. for 2×1 hour and exposed toKodak XAR film with an intensifying screen at −70° C. for 16 hours.

Eleven strongly-hybridizing plaques were picked into PSB and rescreenedas detailed above, to isolate purified plaques. These filters were alsoprobed with ³²P-labelled fragment of the petunia Ht1 cDNA insert(OGR-38), contained in pCGP1805, under low stringency conditions. Lowstringency conditions included prehybridization and hybridization at 42°C. in 20% (v/v) formamide, 1 M NaCl, 10% (w/v) dextran sulphate, 1%(w/v) SDS and washing in 6×SSC, 1% (w/v) SDS (w/v) at 65° C. for 1 hour.

The OGR-38 and p58092.13 probes hybridized with identical plaques. The11 pure plaques were picked into PSB and the plasmid vectors pZL1containing the cDNA clones were rescued using the bacterial strainDH10B(Zip). Plasmid DNA was prepared (Del Sal et al., 1989) and the cDNAinserts were released upon digestion with BamHI and EcoRI. The 11plasmids contained cDNA inserts of between 800 bp and 1 kb. Sequencedata generated from the 5′ region of the cDNA inserts suggested thatnine of these clones were identical. Sequence data were generated fromthe 5′ ends of all nine cDNA inserts and the 3′ end of only one cDNAinsert. The sequence data generated from all clones were compiled toproduce the nucleotide and translated sequence shown as SEQ ID NO:7 andSEQ ID NO:8.

The A. thaliana putative F3′H sequences were compared with the sequencesof the petunia OGR-38 F3′H cDNA clone (SEQ ID NO:1 and SEQ ID NO:2) andwas 64.7% similar to the petunia F3′H cDNA clone, over 745 nucleotides,and 63.7% similar, over 248 amino acids.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

Isolation of a F3′H Genomic Clone From Arabidopsis Thaliana

To isolate a genomic clone of the A. thaliana F3′H gene, a A. thalianaecotype Landsberg erecta genomic DNA library was screened with³²P-labelled p60606.04 fragments. The library was created by cloningpartial MboI-digested genomic DNA between BamHI-digested bacteriophagelambda EMBL4 arms. The primary library, which contained 30,000 clones,was amplified once before screening.

The p60606.04 clone, containing a 1 kb fragment of A. thaliana F3′HcDNA, was digested with BamHI/EcoRI to excise the insert which waspurified using GeneClean (Bio 101). Probe was ³²P-labelled using thenick-translation procedure. (Sambrook et al., 1989). Approximately20,000 plaques were probed at high stringency (50% formamide at 37° C.)and filters were washed in: 2×SSPE; 2×SSPE, 0.1% (w/v) SDS; 0.1×SSPE,all at 65° C. Re-screening was carried out under the same conditions.

DNA was purified from three positive plaques (λTT7-1, λTT7-5 and λTT7-6)and mapped by digestion with EcoRI and EcoRI/SalI. All three clones hadan EcoRI fragment in common. λTT7-1 and λTT7-5 had overlapping but notidentical restriction patterns. A Southern blot of these digests wasprobed as above and, for λTT7-1 and λTT7-5, a common 6.5 kb EcoRI/SalIfragment hybridized. A smaller EcoRI/SalI fragment in λTT7-6 alsohybridized and was presumably at the insert boundary.

EcoRI/SalI fragments from 1TT7-5 were cloned into pBlueScript SK+ and aclone containing the 6.5 kb fragment, designated E-5, was identified byhybridization (as above) and insert size. A restriction map was compiledfor the fragment using EcoRI, SalI, KpnI, HindIII and BglII in variouscombinations, and by hybridization to Southern blots of these digestswith the BamHI/EcoRI insert from the A. thaliana F3′H cDNA clone.

Complete Sequence of Tt7 Genomic Clone

A 6.4 kb BamHI fragment from Tt7-2, containing most of the Tt7 genomicfragment was purified, self-ligated, sonicated, end-repaired,size-fractionated (450 bp to 800 bp) and cloned into SmaI-cut pUC19using standard techniques (Sambrook et al., 1989). Recombinant cloneswere isolated, and plasmid DNA was purified and sequenced using M13-21or M13 reverse sequencing primers. The sequence from overlapping cloneswas combined into one contiguous fragment. The sequence of the ends ofthe Tt7 genomic fragment were also obtained by sequencing with the −21and REV primers. All of the sequences were combined together to obtainthe complete sequence of the 6.5 kb EcoRI/SalI fragment from E-5 (SEQ IDNO:9).

The sequences over the coding region of the arabidopsis Tt7 genomicclone (SEQ ID NO: 10, 11, 12 and 13) were compared with those of thepetunia OGR-38 F3′H cDNA clone (SEQ ID NO: 1 and 2). The arabidopsis Tt7coding region showed 65.4% similarity, over 1066 nucleotides, and 67.1%similarity, over 511 amino acids, to that of the petunia OGR-38 F3′HcDNA clone.

Transformation of a tt7 Arabidopsis Mutant

Preparation of Binary Vector

The EcoRI/SalI fragment from E-5 was cloned into EcoRI/SalI-cut pBI101(Jefferson et al., 1987). Two separate but identical clones wereidentified: pBI-Tt7-2 (FIG. 15) and pBI-Tt7-4. Both clones were used fortransformation of A. tumefaciens.

Plant Transformation

Plasmids pBI-Tt7-2, pBI-Tt74 and pBI101 were transformed intoAgrobacterium strain GV3101 pMP90 by electroporation. Transformants wereselected on medium containing 50 μg/mL kanamycin (and 50 μg/mLgentamycin to select for the resident pMP90).

Plasmid DNA, from four transformant colonies for each clone, wasisolated and digested with EcoRI/SalI, electrophoresed, Southernblotted, and probed with the TT7 cDNA insert. For pBI-Tt7-2 andpBI-Tt74, the expected insert band was identified.

One transformant for each plasmid (i.e.: one control [pBI101 C4], oneeach of the two Tt7 clones [pB1-Tt7-2-3 and pBI Tt7-4-4]) was used tovacuum infiltrate the A. thaliana tt2 mutant line NW88 (4 pots of 10plants each for each construct), using the a method essentially asdescribed by Bechtold et al. (1993).

Seed from each pot was harvested. One hundred mg of seed (approximately5,000) was plated on nutrient medium (described by Haughn andSomerville, 1986) containing 50 μg/mL kanamycin. Kanamycin-resistanttransformants were visible after 7 to 10 days. In the case ofpBI-Tt7-2-3 and pBI-Tt744, a total of 1-1 transformants were isolatedfrom 5 different seed lots (i.e.: pots) and all kanamycin-resistanttransformants were visibly Tt7 in phenotype and exhibited thecharacteristic red/purple anthocyanin pigments at the margins of thecotyledons and at the hypocotyl. A single kanamycin-resistanttransformant was isolated from only one of the four pots of controltransformants and it did not exhibit a “wild-type” M phenotype.

Complementation of tt7 Mutant

These transformants were planted out and grown to maturity andindividually harvested for seed. In each case, for pBI-Tt7-2-3 andpBI-Tt7-4-4 transformants, the seeds were visibly more brown than thepale brown seed of the tt7 mutant plants. The seed from the controltransformant was indistinguishable from the tt7 mutant parent. Theseseed were plated out on nutrient medium and nutrient medium withkanamycin added, and scored for the Tt7 phenotype (red/purpleanthocyanin pigments at the margins of the cotyledons and at thehypocotyl) and kanamycin resistance. The progeny of at least onetransformant for each seed lot was examined, since these were clearlyindependent transformation events.

Without exception, kanamycin-resistant seedlings exhibited the Tt7phenotype while kanamycin-sensitive individuals were tt7. In some cases,kanamycin resistance was weak and variable among a family of seed and itwas difficult to unequivocally determine whether individuals werekanamycin resistant or kanamycin sensitive.

EXAMPLE 24 Isolation of a F3′H cDNA Clone From Rosa hybrida

In order to isolate a Rose F3′H cDNA clone, a Rosa hybrida cv. Kardinalpetal cDNA library was screened with ³²P-labelled fragments of thepetunia Ht1 cDNA clone (OGR-38), contained in pCGP1805, and snapdragonF3′H cDNA clone (sdF3′H), contained in pCGP246.

Construction of a Petal cDNA Library from Rose cv. Kardinal

Total RNA was prepared from the buds of Rosa hybrida cv. Kardinal stage2. At this stage, the tightly closed buds were 1.5 cm high andapproximately 0.9 cm wide with pale pink petals.

Frozen tissue (1-3 g) was ground in liquid nitrogen with a mortar andpestle, placed in 25 mL pre-chilled Buffer A [0.2 M boric acid, 10 mMEDTA (sodium salt) (pH 7.6)] and homogenized briefly. The extract wasmixed on a rotary shaker until it reached room temperature and an equalvolume of phenol/chloroform (1:1 v/v), equilibrated with Buffer A, wasadded. After mixing for a further 10 minutes, the RNA preparation wascentrifuged at 10,000×g for 10 minutes at 20° C. The upper aqueous phasewas retained and the phenol interface re-extracted as above. The aqueousphases were pooled and adjusted to 0.1 M sodium acetate (pH 6.0), 2.5volumes 95% ethanol were added and the mixture was stored at −20° C.overnight.

The preparation was centrifuged at 10,000×g for 10 minutes at 4° C., thepellet dissolved gently in 20 mL Buffer B [25 mM boric acid, 1.25 MmEDTA (sodium salt), 0.1 M NaCl (pH 7.6)] and 0.4 volumes 2-butoxyethanol(2BE) were added. This solution was incubated on ice for 30 minutes. Itwas then centrifuged at 10,000×g for 10 minutes at 0° C. and thesupernatant was carefully collected. After addition of 1.0 volume of 2BEand incubation on ice for a further 30 minutes, the supernatant wasagain centrifuged at 10,000×g for 10 minutes at 0° C. The resultingpellet was gently washed with Buffer A:2BE (1:1 v/v), then with 70%(v/v) ethanol, 0.1 M potassium acetate and finally with 95% ethanol. Thepellet was air dried and dissolved in 1 mL diethyl pyrocarbonate(DEPC)-treated water. This was adjusted to 3 M lithium chloride, left onice for 60 minutes and centrifuged at 10,000×g for 10 minutes at 0° C.The pellet was washed twice with 3 M LiCl and then with 70% ethanol, 0.1M potassium acetate.

The resulting RNA pellet was dissolved in 400 μL DEPC-treated water andextracted with an equal volume phenol/chloroform. The RNA mix was thencentrifuged at 10,000×g for 5 minutes at 20° C., the aqueous phasecollected and made to 0.1 M sodium acetate, and a further 2.5 volumes of95% ethanol were added. After 30 minutes incubation on ice, the mix wascentrifuged at 13,000 rpm (5,000×g) for 20 minutes at 20° C. and the RNApellet resuspended gently in 400 μL DEPC-treated water.

Poly (A)⁺ RNA was selected from the total RNA by Oligotex dT-30 (Takara,Japan) following the manufacturer's protocol. The cDNA was synthesizedaccording to the method in Brugliera et al. (1994) and used to constructa non-directional petal cDNA library in the EcoRI site of λZAPII(Stratagene). The total number of recombinants obtained was 3.5×10⁵.

After transfecting XL1-Blue cells, the packaged cDNA mixture was platedat 50,000 pfu per cm diameter plate. The plates were incubated at 37° C.for 8 hours, and the phage were eluted in 100 mM NaCl, 8 mM MgSO₄, 50 mMTris-HCl pH 8.0, 0.01% (w/v) gelatin (Phage Storage Buffer (PSB))(Sambrook et al., 1989). Chloroform was added and the phage stored at 4°C. as an amplified library. 200,000 pfus of the amplified library wereplated onto NZY plates (Sambrook et al., 1989) at a density of 10,000pfu per 15 cm plate after transfecting XL1-Blue MRF′ cells, andincubated at 37° C. for 8 hours. After incubation at 4° C. overnight,duplicate lifts (labelled as group A and group B) were taken ontoColony/Plaque Screen™filters (DuPont) and treated as recommended by themanufacturer.

Screening of Kardinal cDNA Library for a F3′H cDNA Clone

Prior to hybridization, the duplicate plaque lifts were washed inprewashing solution (50 mM Tris-HCl pH7.5, 1 M NaCl, 1 mM EDTA, 0.1%(w/v) sarcosine) at 65° C. for 30 minutes; stripped in 0.4 M sodiumhydroxide at 65° C. for 30 minutes; then washed in a solution of 0.2 MTris-HCl pH 8.0, 0.1×SSC, 0.1% (w/v) SDS at 65° C. for 30 minutes andfinally rinsed in 2×SSC, 1.0% (w/v) SDS.

The group A filters of the duplicate lifts from the Kardinal cDNAlibrary were screened with ³²P-labelled fragments of an NcoI fragmentfrom pCGPI805 containing the petunia Ht1 (OGR-38) cDNA clone, while thegroup B filters were screened with ³²P-labelled fragments of EcoRI/SspIfragment from pCGP246 containing the snapdragon F3′H clone.

Hybridization conditions included a prehybridization step in 10% (v/v)formamide, 1 M NaCl, 10% (w/v) dextran sulphate, 1% (w/v) SDS at 42° C.for at least 1 hour. The ³²P labelled fragment (2×10⁶ cpm/mL) was thenadded to the hybridization solution and hybridization was continued at42° C. for a further 16 hours. The filters were then washed at 42° C. in2×SSC, 1% (w/v) SDS for 2 hours followed by 1×SSC, 1% (w/v) SDS for 1hour and finally in 0.2×SSC/1% (w/v) SDS for 2 hours. The filters wereexposed to Kodak XAR film with an intensifying screen at −70° C. for 16hours.

Four strongly-hybridizing plaques (R1, R2, R3, R4) were picked into PSBand rescreened to isolate pure plaques. The plasmids contained in theλZAP bacteriophage vector were rescued and digested with EcoRI torelease the cDNA inserts. Clone R1 contained a 1.0 kb insert whileclones R2, R3 and R4 contained inserts of approximately 1.3 kb each.Sequence data were generated from the 3′ and 5′ ends of the R4 cDNAinsert.

The rose R4 putative F3′H sequence was compared with that of the petuniaOGR-38 F3′H sequence. At the nucleotide level, the R4 cDNA clone showed63.2% and 62.1% similarity over 389 nucleotides at the 5′ end and 330nucleotides at the 3′ end, respectively. At the amino acid level, the R4clone showed 65.4% and 73.9% similarity over 130 amino acids at the 5′end and 69 amino acids at the 3′ end, respectively. Based on the highsequence similarity of the Rose R4 cDNA clone to that of the petuniaF3′H cDNA clone (OGR-38), a corresponding “full-length” cDNA clone wasisolated, as described in Example 25, below.

EXAMPLE 25 Isolation of a Full-Length Rose F3′H cDNA

In order to isolate a full-length” F3′H cDNA clone from Rose, the Rosahybrida cv Kardinal petal cDNA library described in Example 24 wasscreened with ³²P-labelled fragments of the rose R4 cDNA clone,described above.

A total of 1.9×10⁶ pfus of the amplified library were plated onto NZYplates at a density of 100,000 pfus per 15 cm diameter plate aftertransfecting XL1-Blue MRF′ cells, and incubated at 37° C. for 8 hours.After incubation at 4° C. overnight, duplicate lifts were taken ontoColony/Plaque Screens filters (DuPont) and treated as recommended by themanufacturer.

Screening of Kardinal cDNA Library for Full-Length P3′H cDNA Clones

Prior to hybridization, the duplicate plaque lifts were treated asdescribed in Example 24.

The duplicate lifts from the Kardinal cDNA library were screened with³²P-labelled fragments of an EcoRI fragment from the rose R4 cDNA clone.

Hybridization conditions included a prehybridization step in 50% (v/v)formamide, 1 M NaCl, 10% (w/v) dextran sulphate, 1% (w/v) SDS at 42° C.for at least 1 hour. The ³²P labelled fragment of the rose R4 cDNA clone(1×10⁶ cpm/mL) was then added to the hybridization solution andhybridization was continued at 42° C. for a further 16 hours. Thefilters were then washed in 2×SSC, 1% (w/v) SDS at 42° C. for 2×1 hourand exposed to Kodak XAR film with an intensifying screen at −70° C. for16 hours.

Seventy-three strongly-hybridizing plaques (1-73) were picked into 1 mLof PSB and stored at 4° C. overnight. 100 μL of each was then aliquotedinto a microtitre tray as an ordered array.

XL1-Blue MRF′ cells were added to 10 mL of molten NZY top agar, pouredonto NZY plates (15 cm diameter) and allowed to set. A replica platingdevice was used to transfer the 73 phage isolates in an ordered arrayonto the NZY plate previously inoculated with the XL1-Blue MRF′ cells.After incubation at 37° C. for 6 hours followed by 4° C. overnight,triplicate lifts (arrays 1, 2 and 3) were taken onto Colony/PlaqueScreen™filters (DuPont) and treated as recommended by the manufacturer.

Prior to hybridization, the duplicate plaque lifts were treated asdescribed in Example 24.

The 3 arrays were screened with ³²P-labelled fragments of a) anEcoRI/SalI fragment covering the 5′ end of the rose R4 cDNA clone, b) anEcoRI/ClaI fragment covering the 5′ end of the rose R4 cDNA clone or c)an EcoRI fragment of the entire rose R4 cDNA clone using thehybridisation and washing conditions described above, except that thefinal wash was in 0.1×SSC, 0.1% (w/v) SDS at 65° C. for 30 minutes. Thefilters were exposed to Kodak XAR film with an intensifying screen at−70° C. for 16 hours.

All 73 plaques hybridised with the full R4 cDNA clone (EcoI fragment)whilst a total of only 17 hybridised with the 5′ end of the R4 cDNAclone (either EcoRI/SalI or the EcoRI/ClaI fragments). The 17 phageisolates were rescreened as described above to isolate purified plaques.Pure plaques were obtained from 9 out of the 17 (2, 4, 26, 27, 34, 38,43, 44, 56). The plasmids contained in the λZAP bacteriophage vectorwere rescued and the sizes of the cDNA inserts were determined using anEcoRI digestion. The cDNA inserts ranged from 0.9 kb to 1.9 kb. Of thenine, only #34 (named pCGP2158) and #38 (named pCGP2159) contained cDNAinserts of approximately 1.9 kb. Sequence data were generated from the3′ and 5′ ends of the cDNA inserts and showed that clones #34 and #38represented the same gene.

The complete sequence of the rose cDNA clone (#34) contained in theplasmid pCGP2158 was determined by compilation of sequence fromdifferent pUC18 subclones obtained using standard procedures for thegeneration of randomly-overlapping clones (Sambrook et al., 1989). Thesequence (SEQ ID NO: 14) contained an open reading frame of 1696 baseswhich encodes a putative polypeptide of 520 amino acids (SEQ ID NO: 15).

The nucleotide and predicted amino acid sequences of the rose F3′H #34cDNA clone (SEQ ID NO: 14 and SEQ ID NO: 15) were compared with those ofthe petunia OGR-38 F3′H cDNA clone (SEQ ID NO: 1 and SEQ ID NO:2) andthe snapdragon sdF3′H clone (SEQ ID NO:3 and SEQ ID NO:4). The rose F3′H#34 cDNA clone showed 64.7% similarity, over 1651 nucleotides, and 72.7%similarity, over 509 amino acids, to that of the petunia OGR-38 cDNAclone, and 67.2% similarity, over 1507 nucleotides, and 68.9 similarity,over 502 amino acids, to that of the snapdragon sdF3′H clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 26 Stable Expression of the Rose F3′H cDNA Clone (#34) inPetunia Petals—Complementation of a ht1/ht1 Petunia Cultivar Preparationof pCGP2166

Plasmid pCGP2166 (FIG. 16) was constructed by cloning the cDNA insertfrom pCGP2158 in a “sense” orientation behind the Mac promoter (Comai etal., 1990) of pCGP293 (Brugliera et al., 1994). The plasmid pCGP2158 wasdigested with EcoRI to release the cDNA insert. The overhanging 5′ endswere filled in using DNA polymerase (Klenow fragment) (Sambrook et al.,1989). The cDNA fragment was isolated and ligated with filled in BamHIends of the pCGP293 binary vector. Correct insertion of the fragment inpCGP2166 was established by restriction enzyme analysis of DNA isolatedfrom gentamycin-resistant transformants.

The binary vector pCGP2166 was introduced into A. tumefaciens strainAGL0 cells, as described in Example 9. The pCGP2166/AGL0 cells were thenused to transform Skr4×SW63 petunia plants (also described in Example9), to test for stable expression and activity of the enzyme encoded bythe gene corresponding to the rose #34 cDNA clone.

EXAMPLE 27 Transgenic Plant Phenotype Analysis

pCGP2166 in Skr4×SW63

The expression of the introduced rose F3′H cDNA in the Skr4×SW63 hybridhad a marked effect on flower colour. The stamen tissue of thenon-transgenic control is white, whereas the same tissue in most of thetransgenic plants was pink. In addition, expression of the rose F3′HcDNA in the Skr4×SW63 hybrid conferred a dark pink hue (RHSCC# 64C and74C) to the corolla, which is normally pale lilac (RHSCC# 75C). Thecolour codes are taken from the Royal Horticultural Society's ColourChart (RHSCC). They provide an alternative means by which to describethe colour phenotypes observed. The designated numbers, however, shouldbe taken only as a guide to the perceived colours and should not beregarded as limiting the possible colours which may be obtained.

Acid-hydrolysed floral extracts (see Example 11) were run in a Forestalsolvent system (HOAc:water:HCl; 30:10:3) (Markham, 1982). The 3′hydroxylated flavonoids, peonidin and quercetin, were readily detectedin the petal limbs of the transgenic plants. Only kaempferol and a smallamount of malvidin were detected in the non-transgenic Skr4×SW63control.

The accumulation of the 3′-hydroxylated anthocyanidin, peonidin and theflavonol, quercetin, in the petals of the transgenic Skr4×SW63/pCGP2166plants correlated with the pink and dark pink colours observed in thepetals of the same plants.

Preparation of pCGP2169

The binary construct pCGP2169 (FIG. 17) was prepared by cloning the cDNAinsert from pCGP2158 in a “sense” orientation between the CaMV35Spromoter (Franck et al., 1980; Guilley et al., 1982) and ocs terminator(De Greve et al., 1982). The plasmid pCGP1634 contained a CaMV35Spromoter, β-glucuronidase (GUS) reporter gene encoded by the E. coliuidA locus (Jefferson et al., 1987) and ocs terminator region in a pUC19vector. The plasmid pCGP2158 was digested with NcoI/XbaI to release thecDNA insert. The plasmid pCGP1634 was also digested with NcoI/XbaI torelease the backbone vector containing the CaMV35S promoter and the ocsterminator. The fragments were isolated and ligated together to producepCGP2167. The plasmid pCGP2167 was subsequently digested with PvuII torelease the expression cassette containing the CaMV35S promoter, therose F3′H cDNA clone and the ocs termintor. This expression cassettefragment was isolated and ligated with SmaI ends of pWTT2132 binaryvector (DNA Plant Technology Corporation; Oakland, Calif.) to producepCGP2169 (FIG. 17).

The binary vector pCGP2169 was introduced into A. tumefaciens strainAGL0 cells, as described in Example 9. The pCGP2169/AGL0 cells are usedto transform rose plants, to reduce the amount of 3′-hydroxylatedflavonoids.

EXAMPLE 28 Isolation of a Putative F3′H cDNA Done From Chrysanthemum

In order to isolate a chrysanthemum F3′H cDNA clone, a chrysanthemum cv.Red Minstral petal cDNA library was screened with ³²P-labelled fragmentsof the petunia Ht1 cDNA clone (OGR-38), contained in pCGP1805.

Construction of a Petal cDNA Library From Chrysanthemum cv. Red Minstral

Total RNA was prepared from the petals (stages 3 to 5) of chrysanthemumcv. Red Minstral using Trizol™reagent (Life Technologies) (Chomczynskiand Sacchi, 1987) according to the manufacturer's recommendations.Poly(A)⁺ RNA was enriched from the total RNA, using a mRNA isolation kit(Pharmacia) which relies on oligo-(dT) affinity spun-columnchromatography.

A Superscript™ cDNA synthesis kit (Life Technologies) was used toconstruct a petal cDNA library in ZipLox using 5 μg of poly(A)+ RNAisolated from stages 3 to 5 of Red Minstral as template.

30,000 pfus of the library were plated onto LB plates (Sambrook et al.,1989) at a density of 3,000 pfus per 15 cm plate after transfectingY1090r-, and incubated at 37° C. for 16 hours. After incubation at 4° C.for one hour, duplicate lifts were taken onto Hybond N+™filters(Amersham) and treated as recommended by the manufacturer.

Screening of the Red Minstral cDNA Library

The duplicate lifts from the Red Minstral petal cDNA library werescreened with ³²P labelled fragments of the 1.8 kb Asp718/BamHI insertfrom pCGP1805.

Hybridization conditions included a prehybridization step in 1 mM EDTA(pH8.0), 0.5MNa₂HPO₄ (pH7.2), 7% (w/v) SDS (Church and Gilbert, 1984) at65° C. for at least 1 hour. The ³²P-labelled fragments (1×10⁶ cpm/mL)were then added to the hybridization solution and hybridization wascontinued at 65° C. for a further 16 hours. The filters were then washedin 2×SSC, 0.1% (w/v) SDS at 65° C. for 2×1 hour and exposed to KodakBioMax™ film with an intensifying screen at −70° C. for 48 hours.

Eight strongly-hybridizing plaques were picked into PSB (Sambrook etal., 1989). Of these, 2 (RM6i and RM6ii) were rescreened to isolatepurified plaques, using the hybridization conditions as described forthe initial screening of the cDNA library. The plasmids contained in theλZipLox bacteriophage vector were rescued according to themanufacturer's protocol and sequence data was generated from the 3′ and5′ ends of the cDNA inserts. The partial sequences of the RM6i and RM6iicDNA inserts were compared with the complete sequence of the petuniaOGR-38 F3′H cDNA clone. The RM6i cDNA clone showed relatively highsequence similarity with that of the petunia OGR-38 cDNA clone, and wasfurther characterised.

The RM6i cDNA insert contained in pCHRM1 was released upon digestionwith EcoRI and was approximately 1.68 kb. The complete sequence of RM6icDNA clone (SEQ ID NO: 16) contained in the plasmid pCHRM1 wasdetermined by compilation of sequence from subclones of the RM6i cDNAinsert.

The nucleotide and predicted amino acid sequences of the chrysanthemumRM6i cDNA insert (SEQ ID NO: 16 and SEQ ID NO: 17) were compared withthose of the petunia OGR-38 F3′H cDNA clone (SEQ ID NO:1 and SEQ IDNO:2). The sequence of the chrysanthemum RM6i cDNA insert showed 68.5%similarity, over 1532 nucleotides, and 73.6% similarity, over 511 aminoacids, to that of the petunia OGR-38 F3′H cDNA clone. An alignment ofthe petunia, carnation, snapdragon, arabidopsis, rose, chrysanthemum andtorenia sequences, all of which are disclosed in this specification, andvarious summaries of comparisons of sequence similarities among thenucleotide and corresponding amino acid sequences, can be found in Table7 and in Tables 8, 9, 10, 11 and 12, respectively. These Tables are inExample 34, at the end of the specification.

Construction of pLN85 (Antisense Binary)

A plasmid designated pLN84 was constructed by cloning the RM6i cDNAinsert from pCHRM1 in the “antisense” orientation behind the completeCaMV35S promoter contained in pART7 (Gleave 1992). The plasmid pCHRM1was digested with NotI to release the cDNA insert. The RM6i cDNAfragment was blunt-ended using T4 DNA polymerase (Sambrook et al., 1989)and purified, following agarose gel electrophoresis and GELase(Epicentre Technologies). The purified fragment was ligated with 5=n1ends of the pART7 shuttle vector to produce pLN84. The plasmid pLN84 wassubsequently digested with NotI to release the expression cassettecontaining CaMV35S: RM6i cDNA: ocs. The expression cassette was isolatedas a single fragment and ligated with NotI ends of the pART27 binaryvector (Gleave, 1992) to produce pLN85 (FIG. 18). Correct insertion ofthe fragment was established by restriction enzyme analysis of DNAisolated from streptomycin-resistant E. coli transformants.

The binary vector pLN85 is introduced into chrysanthemum plants viaAgrobacterium-mediated transformation, as described in Ledger et al,1991), to reduce the amount of 3′-hydroxylated flavonoids.

EXAMPLE 29 Isolation of a Putative F3′H cDNA Clone From Toreniafournieni

In order to isolate a torenia F3′H cDNA clone, the petunia Ht1-linkedF3′H cDNA clone (OGR-38), contained in pCGP1805, was used to screen aTorenia founieri cv. Summer Wave petal cDNA library, under lowstringency conditions.

Construction of Torenia Founieri cv. Summer Wave Petal cDNA Library

A directional petal cDNA library was prepared from Summer Wave flowers,essentially as described in Example 4.

Screening of Summer Wave Petal cDNA Library

Lifts of a total of 200,000 of the amplified Summer Wave petal cDNAlibrary were screened with DIG-labelled fragments of the 1.8 kb OGR-38cDNA insert from pCGPI805. A DIG DNA labelling and detection kit fromBoehringer-Mannheim was used according to the manufacturer'srecommendations.

Hybridizations were carried out in 30% (v/v) formamide. 5×SSC, 1% (w/v)SDS at 37° C. for 16 hours. The filters were then washed in 5×SSC, 1%(w/v) SDS at 65° C. for 1 hour.

The signals were visualized following the protocol of the DIG DNAlabelling and detection kit.

Twelve strongly-hybridizing plaques were picked into PSB and rescreenedto isolate pure plaques. The plasmids contained in the λZAPIIbacteriophage vector were rescued and digested with EcoRI/XhoI torelease the cDNA inserts. Most of the twelve clones contained cDNAinserts of approximately 1.8 kb. One clone, THT52, contained the longest5′ non-coding-region sequence. The complete sequence of the torenia cDNAclone (THT52), contained in the plasmid pTHT52, was determined bycompilation of sequence from different pUC18 subclones obtained usingstandard procedures for the generation of randomly-overlapping clones(Sambrook et al., 1989). The sequence (SEQ ID NO: 18) contained an openreading frame of 1524 bases which encodes a putative polypeptide of 508amino acids (SEQ ID NO: 19).

The nucleotide and predicted amino acid sequences of the torenia THT52cDNA clone (SEQ ID NO:18 and SEQ ID NO: 19) were compared with those ofthe petunia OGR-38 F3′H cDNA clone (SEQ ID NO:1 and SEQ ID NO:2). Thetorenia THT52 cDNA clone showed 63.6% similarity, over 1694 nucleotides,and 67.4% similarity, over 515 amino acids, to that of the petuniaOGR-38 cDNA clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 30 The F3′H Assay of the Torenia THT cDNA Clone Expressed inYeast Construction of pYTHT6

The plasmid pYTHT6 (FIG. 19) was constructed by cloning the cDNA insertfrom pTHT6 in a “sense” orientation behind the yeastglyceraldehyde-3-phosphate dehydrogenase promoter of pYE22m (Tanaka etal., 1988). The plasmid pTHT6 contained the THT6 cDNA clone. THT6 isidentical to THT52, except that its 5′ non-coding region is 75 bpshorter.

The 1.7 kb THT6 cDNA insert was released from the plasmid pTHT6 upondigestion with EcoRI/XhoI. The THT6 cDNA fragment was isolated, purifiedand ligated with E=R1/91 ends of pYE22m to produce pYTHT6.

Yeast transformation, preparation of yeast extracts and the F3′H assayare described in Example 6.

F3′H activity was detected in extracts of G1315/pYTHT6, but not inextracts of non-transgenic yeast. From this it was concluded that theTHT6 cDNA insert contained in pYTHT6, encoded a F3′H.

EXAMPLE 31 Isolation of a Putative F3′H cDNA Clone From Pharbitis Nil(Japanese Morning Glory)

In order to isolate a morning glory F3′H cDNA clone, the petuniaHt1-linked F3′H cDNA clone (OGR-38), contained in pCGP1805, was used toscreen a Japanese morning glory petal cDNA library, under low stringencyconditions.

Construction of Japanese Morning Glory Petal cDNA Library

The petal cDNA library from young petals of Pharbitis nil (Japanesemorning glory) was obtained from Dr Iida (National Institute of BasicBiology, Japan).

Screening of Japanese Morning Glory Petal cDNA Library

Lifts of a total of 200,000 of the amplified Japanese morning glorypetal cDNA library were screened with DIG-labelled fragments of the 1.8kb OGR-38 cDNA insert from pCGPI805. A DIG DNA labelling and detectionkit from Boehringer-Manaheim was used according to the manufacturer'srecommendations.

Hybridizations were carried out in 30% (v/v) formamide, 5×SSC, 1% (w/v)SDS at 37° C. for 16 hours. The filters were then washed in 5×SSC, 1%(w/v) SDS at 65° C. for 1 hour. The signals were visualized followingthe protocol of the DIG DNA labelling and detection kit.

Twenty strongly-hybridizing plaques were picked into PSB and rescreenedto isolate pure plaques. The plasmids contained in the λZAPIIbacteriophage vector were rescued and digested with EcoRI/XhoI torelease the cDNA inserts. One clone (MHT85) contained a 1.8 kb insert.The complete sequence of the Japanese morning glory cDNA clone (MHT85)(SEQ ID NO:20), contained in the plasmid pMHT85, was determined bycompilation of sequence from different pUC18 subclones obtained usingstandard procedures for the generation of randomly-overlapping clones(Sambrook et al., 1989). The MHT85 sequence appears to be 5 bases shortof “full-length”.

The nucleotide and predicted amino acid sequences of the Japanesemorning glory MHT85 cDNA clone (SEQ ID NO:20 and SEQ ID NO:21) werecompared with those of the petunia OGR-38 F3′H cDNA clone (SEQ ID NO:1and SEQ ID NO:2). The Japanese morning glory MHT85 cDNA clone showed69.6% similarity, over 869 nucleotides, and 74.8% similarity, over 515amino acids, to that of the petunia OGR-38 cDNA clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 32 Isolation of a Putative F3′H cDNA Done From Gentiana Triflora

In order to isolate a gentian F3′H cDNA clone, the petunia Ht1-linkedF3′H cDNA clone (OGR-38), contained in pCGP1805, was used to screen aGentiana triflora Pall. var japonica Hara petal cDNA library, under lowstringency conditions.

Construction of Gentian Petal cDNA Library

A petal cDNA library was prepared from Gentiana triflora Pall. varjaponica Hara flowers, as described by Tanaka et al., 1996.

Screening of Gentian Petal cDNA Library

Lifts of a total of 200,000 of the amplified gentian petal cDNA librarywere screened with DIG-labelled fragments of the 1.8 kb OGR-38 cDNAinsert from pCGP1805. A DIG DNA labelling and detection kit fromBoehringer-Mannheim was used according to the manufacturer'srecommendations.

Hybridizations were carried out in 30% (v/v) formamide, 5×SSC, 1% (w/v)SDS at 37° C. for 16 hours. The filters were then washed in 5×SSC, 1%(w/v) SDS at 65° C. for 1 hour. The signals were visualized followingthe protocol of the DIG DNA labelling and detection kit.

Fifteen strongly-hybridizing plaques were picked into PSB and rescreenedto isolate pure plaques. The plasmids contained in the λZAPIIbacteriophage vector were rescued and digested with EcoRI/XhoI torelease the cDNA inserts. One clone (GHT13) contained a 1.8 kb insert.The sequence of the partial gentian cDNA clone (GHT13) (SEQ ID NO:22),contained in the plasmid pGHT13, was determined by compilation ofsequence from different pUC 18 subclones obtained using standardprocedures for the generation of randomly-overlapping clones (Sambrooket al., 1989).

The nucleotide and predicted amino acid sequences of the gentian GHT13cDNA clone (SEQ ID NO:22 and SEQ ID NO:23) were compared with those ofthe petunia OGR-38 F3′H cDNA clone. The gentian GHT13 cDNA clone showed68.3% similarity, over 1519 nucleotides, and 71.8% similarity, over 475amino acids, to that of the petunia OGR-38 cDNA clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

EXAMPLE 33 Isolation of Putative F3′H cDNA Clone From Lisianthus

In order to isolate a lisianthus F3′H cDNA clone, the petunia Ht1-linkedF3′H cDNA clone (OGR-38), contained in pCGP1805, was used to screen alisianthus petal cDNA library, under low stringency conditions.

Construction and Screening of Lisianthus Petal cDNA Library

10,000 pfus of a lisianthus petal cDNA library described by Davies etal. (1993) and Markham and Offman (1993) were plated onto LB plates(Sambrook et al., 1989) at a density of 3,000 pfus per 15 cm plate aftertransfecting Y1090r-, and incubated at 37° C. for 16 hours. Afterincubation at 4° C. for one hour, duplicate lifts were taken onto HybondN+™filters (Amersham) and treated as recommended by the manufacturer.

The duplicate lifts from the lisianthus line #54 petal cDNA library werescreened with ³²P labelled fragments of the 1.8 kb Asp718/BamHI insertfrom pCGP1805.

Hybridization conditions included a prehybridization step in 1 mM EDTA(pH8.0), 0.5MNa₂HPO₄ (pH7.2), 7% (w/v) SDS (Church and Gilbert, 1984) at55° C. for at least 1 hour. The ³²P-labelled fragments (1×10⁶ cpm/mL)were then added to the hybridization solution and hybridization wascontinued at 55° C. for a further 16 hours. The filters were then washedin 2×SSC, 0.1% (w/v) SDS at 55° C. for 2×15 minutes, and exposed toKodak BioMax™ film with an intensifying screen at −70° C. for 18 hours.

Twelve strongly-hybridizing plaques were picked into PSB (Sambrook etal., 1989) and rescreened to isolate purified plaques, using thehybridization conditions as described for the initial screening of thecDNA library. Sequence data were generated from the 3′ and 5′ ends ofthe cDNA inserts of four clones.

Based on sequence comparisons, pL3-6 showed similarity with the petuniaOGR-38 F3′H cDNA clone and was further characterised.

The 2.2 kb cDNA insert, contained in pL3-6, was subsequently found tocontain 3 truncated cDNA clones, the longest (L3-6) having high sequencesimilarity to the petunia OGR-38 cDNA sequence. The sequence of thisL3-6 partial cDNA clone contained in the plasmid pL3-6 was determined bycompilation of sequence from subclones of the L3-6 cDNA insert (SEQ IDNO:24).

The nucleotide and predicted amino acid sequences of the lisianthus L3-6cDNA clone (SEQ ID NO:24 and SEQ ID NO:25) were compared with those ofthe petunia OGR-38 F3′H cDNA clone (SEQ ID NO: 1 and SEQ ID NO:2). Thesequence of the lisianthus L3-6 cDNA clone showed 71.4% similarity, over1087 nucleotides, and 74.6% similarity, over 362 amino acids, to that ofthe petunia OGR-38 F3′H cDNA clone.

An alignment of the petunia, carnation, snapdragon, arabidopsis, rose,chrysanthemum and torenia sequences, all of which are disclosed in thisspecification, and various summaries of comparisons of sequencesimilarities among the nucleotide and corresponding amino acidsequences, can be found in Table 7 and in Tables 8, 9, 10, 11 and 12,respectively. These Tables are in Example 34, at the end of thespecification.

Further investigation of the remaining clones isolated from thescreening of the lisianthus library identified another putative F3′HcDNA clone (L3-10), contained in the plasmid pL3-10. The L3-10 cDNAinsert is approximately 1.8 kb and appears to represent a “full-length”clone.

EXAMPLE 34 Alignments and Comparisons Among Nucleotide and Amino AcidSequences Disclosed Herein

Multiple sequence alignments were performed using the ClustalW programas described in Example 3. Table 7 (below) provides a multiple sequencealignment of the predicted amino acid sequences of petunia OGR-38 (A);carnation (B); snapdragon (C); arabidopsis Tt7 coding region (D); rose(E) chrysanthemum (F); torenia (G); morning glory (H); gentian (partialsequence) (I); lisianthus (partial sequence) (J) and the petunia 651cDNA (K). Conserved amino acids are shown in bolded capital letters andare boxed and shaded. Similar amino acids are shown in capital lettersand are only lightly shaded, and dissimilar amino acids are shown inlower case letters.

Nucleotide and amino acid sequences of the F3′H cDNA clones from theabove mentioned species and the coding region of the genomic clone fromarabidopsis were compared using the LFASTA program, as described inExample 3. Summaries of similarity comparisons are presented in Tables 8to 12, below.

TABLE 8 Percentage of sequence similarity between F3′H sequence ofPetunia OGR-38 and F3′H sequences from other species and other P450molecules Number of % similarity to OGR-38/ % similarity to OGR-38/Number of amino acids no. nt no. aa Species/Clone nucleotides (nt) (aa)(area of similarity) (area of similarity) Petunia OGR-38 1789nt 512aaSnapdragon 1711nt 512aa 69.0%/1573nt 72.2%/507aa F3′H cDNA (19-1578)(1-504) Arabidopsis partial 971nt 270aa 64.7%/745nt 63.7%/248aa F3′HcDNA (854-1583) (269-510) Arabidopsis Tt7 coding 1774nt 513aa65.4%/1066nt 67.1%/511aa region Carnation 1745nt 496aa 67.3%/1555nt71.5%/488aa F3′H cDNA (28-1571) (17-503) Rose 1748nt 513aa 64.7%/1651nt72.7%/509aa F3′H cDNA (56-1699) (7-510) Gentian 1667nt 476aa68.3%/1519nt 71.8%/475aa partial F3′H cDNA (170-1673) (40-510) MorningGlory 1824nt 517aa 69.6%/869nt 74.8%/515aa F3′H cDNA (60-1000) (3-510)Chrysanthemum 1660nt 508aa 68.5%/1532nt 73.6%/511aa F3′H cDNA (50-1580)(1-510) Lisianthus 1214nt 363aa 71.4%/1087nt 74.6%/362aa partial F3′HcDNA (520-1590) (160-510) Torenia 1815nt 508aa 63.6%/1694nt 67.4%/515aaF3′H cDNA (90-1780) (1-510) Petunia Hf1 1812nt 508aa 58.9%/1471nt49.9%/513aa cDNA (29-1474) (1-511) Petunia Hf2 1741nt 508aa 58.9%/1481nt49.1%/511aa cDNA (37-1498) (3-510) Petunia 651 1716nt 496aa 53.5%/1284nt38.0%/502aa cDNA (50-1309) (7-503) Mung Bean 1766nt 505aa 56.0%/725nt29.2%/511aa C4H cDNA (703-1406) (1-503)

TABLE 9 Percentage of sequence similarity between F3′H sequence ofSnapdragon and F3′H sequences from other species and other P450molecules Number of Number of % similarity to % similarity to Species/nucleotides amino snapdragon/ snapdragon/ Clone (nt) acids (aa) no. ntno. aa Snapdragon 1711nt 512aa Petunia 1789nt 512aa 69.0%/1573nt72.2%/507aa OGR-38 F3′H cDNA Arabidopsis 971nt 270aa 64.5%/740nt60.4%/240aa partial F3′H cDNA Carnation 1745nt 496aa 66.7%/1455nt68.4%/487aa F3′H cDNA Torenia 1815nt 508aa 67.6%/1603nt 70.3%/505aa F3′HcDNA Rose 1748nt 513aa 67.2%/1507nt 68.9%/502aa F3′H cDNA Petunia Hf11812nt 508aa 57.3%/1563nt 49.3%/491aa cDNA Petunia Hf2 1741nt 508aa57.7%/1488nt 47.8%/508aa cDNA Petunia 651 1716nt 496aa 54.4%/1527nt39.0%/493aa cDNA Mung Bean 1766nt 505aa 50.6%/1344nt 32.0%/490aa C4HcDNA

TABLE 10 Percentage of sequence similarity between F3′H sequence ofArabidopsis and F3′H sequences from other species and other P450molecules % % Number of Number of similarity to similarity to Species/nucleotides amino Arabidopsis/ Arabidopsis/ Clone (nt) acids (aa) no. ntno. aa Arabidopsis  971nt 270aa Petunia OGR-38 1789nt 512aa 64.7%/745nt63.7%/248aa F3′H cDNA Snapdragon 1711nt 512aa 64.5%/740nt 60.4%/240aaF3′H cDNA Carnation 1745nt 496aa 64.7%/782nt 60.6%/241aa F3′H cDNA Rose1748nt 513aa 68.5%/739nt 63.7%/248aa F3′H cDNA Petunia 651 1716nt 496aa57.0%/521nt 40.5%/227aa cDNA Petunia Hf1 1812nt 508aa 58.2%/632nt46.5%/243aa cDNA Petunia Hf2 1741nt 508aa 57.4%/632nt 46.1%/243aa cDNA

TABLE 11 Percentage of sequence similarity between F3′H sequence of Roseand F3′H sequences from other species and other P450 molecules Number ofNumber of nucleotides amino % similarity to Rose/ % similarity to Rose/Species/Clone (nt) acids (aa) no. nt no. aa Rose 1748bp 513aa PetuniaOGR-38 1789bp 512aa 64.7%/1651nt 72.7%/509aa F3′H cDNA Snapdragon 1711bp512aa 67.2%/1507 68.9%/502aa F3′H cDNA Carnation 1745bp 496aa67.4%/1517nt 72.6%/486aa F3′H cDNA Arabidopsis  971bp 270aa 68.5%/739nt63.7%/248aa partial F3′H cDNA Petunia 651 1716bp 496aa 53.1%/1182nt37.8%/502aa cDNA Petunia Hf1 1812bp 506aa   57%/1366nt 49.9%/503aa cDNAPetunia Hf2 1741bp 508aa 57.3%/1331nt 49.1%/505aa cDNA Mung Bean 1766bp505aa 52.4%/1502nt 32.0%/510aa C4H cDNA

TABLE 12 Percentage of sequence similarity between coding region ofArabidopsis tt7 genomic sequence and F3′H cDNA sequences from otherspecies and other P450 molecules % similarity Number of Number of %similarity to to nucleotides amino Arabidopsis Arabidopsis Species/Clone(nt) acids (aa) tt7/no. nt tt7/no. aa Arabidopsis 1774nt 513aa Tt7coding region Petunia 1789nt 512aa 65.4%/1066nt 67.1%/511aa OGR-38 F3′HcDNA Snapdragon 1711nt 512aa 62.7%/990nt 64.9%/504aa F3′H cDNA Carnation1745nt 496aa 63.2%/1050nt 65.9%/495aa F3′H cDNA Rose 1748nt 513aa65.5%/1076nt   68%/512aa F3′H cDNA Petunia 651 1716nt 496aa 56.5%/990nt36.5%/502aa cDNA Petunia Hf1 1812nt 506aa 56.8%/995nt 47.5%/509aa F3′HcDNA Petunia Hf2 1741nt 508aa 55.2%/1063nt 46.8%/509aa F3′H cDNA

Those skilled in the art, will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

-   Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and    Lipman, D. J. J. Mol. Biol. 215: 403410, 1990.-   Ashikari, T., Kiuchi-Goto, N., Tanaka, Y., Shibano, Y., Amachi, T.,    and Yoshizumi, H. Appl. Microbiol. Biotechnol. 30: 515-520, 1989.-   Baird, W. V. and Meagher, R. B. EMBO J. 6, 3223-3231, 1987.-   Bechtold, N., Ellis, J. and Pelletier, G. C. R. Acad. Sci. Paris,    Sciences de la vie 316: 1194-1199, 1993.-   Bethesda Research Laboratories. BRL pUC host: E. coli DH5α™    competent cells. Bethesda Res. Lab. Focus. 8(2): 9, 1986.-   Brugliera, F., Holton, T. A., Stevenson, T. W., Farcy, E., Lu, C-Y    and Cornish, E. C. Plant J. 5(1): 81-92, 1994.-   Church, G. M. and Gilbert, W. PNAS USA, 81: 1991-1995.-   Chomczynski, P. and Sacchi, N. Anal Biochem. 162: 156-159.-   Comai, L., Moran, P. and Maslyar, D., Plant Mol. Biol. 15: 373-381,    1990.-   Cornu, A., Farcy, E., Maizonnier, D., Haring, M., Veerman, W. and    Gerats, A. G. M., In: Genetic maps—Locus maps of complex genomes.    5th edition, Stephen J. O'Brien (ed.), Cola Spring Harbor Laboratory    Press, USA, 1990.-   Davies et al., Plant Science, 95: 67-77, 1993.-   D'Alessio et al., Focus, 14: 76-79, 1992-   De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M    and Schell, J. J. Mol Appl Genet. 1: 499-511.-   Dellaporta, S. J., Wood, J. and Hick, J. B., Plant Mol. Biol. Rep.    1: 19-21, 1983.-   Del Sal, G., Manfioletti, G. and Schneider, C. Biotechniques, 7(5):    514-519, 1989.-   Doodeman, M., Gerats, A. G. M., Schram, A. W., De Vlaming, P. and    Bianchi, F., Theor. Appl. Genet. 67: 357-366, 1984.-   Dooner, H. K., Robbins, T. R. and Jorgensen, R. A. Ann. Rev. Genet.    25: 173-199, 1991.-   Ebel, J. and Hahlbrock, K., In: The Flavonoids: Advances in Research    Since 1980. Harbourne, J. B. (ed.), Academic Press, New York, USA,    641-679, 1988.-   Forkmann, G. and Stotz, G. Z. Naturforsch. 36c:411-416, 1981.-   Forkmann, G. Plant Breeding 106: 1-26, 1991.-   Franck, A., Guilley, H., Jonard, G. Richards, K. and Hirth, L. Cell,    21, 285-294, 1980.-   Frohman, M. A., Dush, M. K., Martin, G. R. Proc. Natl. Acad. Sci.    USA 85: 8998-9002, 1988.-   Gamborg, O. L., Miller, R. A. and Ojima, K., Exp. Cell Res. 50:    151-158, 1968.-   Garfinkel, D. J. and Nester, E. W. J. Bacteriol. 144:732-743, 1980.-   Gleave, A. P. Plant Molecular Biology 20: 1203-1207, 1992.-   Guilley, H., Dudley, R. K., Jonard, G., Balazs, E. and    Richards, K. E. Cell, 30, 763-773, 1982.-   Hahlbrock, K. and Grisebach, H., Annu. Rev. Plant Physiol. 30:    105-130, 1979.-   Hanahan, D., J. Mol. Biol. 166: 557, 1983.-   Haughn, G. W. and Somerville, C. Molecular and General Genetics 204:    430-434, 1986.-   Holton, T. A., Brugliera, F. Lester, D. R., Tanaka, Y., Hyland, C.    D., Menting, J. G. T., Lu, C., Farcy, E., Stevenson, T. W. and    Cornish, E. C., Nature, 366, 276-279, 1993.-   Holton, T. A. and Cornish, E. C. Plant Cell, 7: 1071-1083, 1995.-   Inoue, H., Nojima, H. and Okayama, H. Gene, 96: 23-28, 1990.-   Ito, H., Fukuda, Y., Murata, K. and Kimura, A. J. Bacteriol. 153:    163-168, 1983.-   Jefferson, R. A. Plant Mol. Biol. Rep. 5: 387-405, 1987.-   Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. EMBO J. 6:    3901-3907, 1987-   Koornneef, M, Luiten, W., de Vlaming, P. and Schram, A. W.    Arabidopsis Information Service 19: 113-115, 1982.-   Kozak, M. J. Cell. Biol. 108: 229, 1989.-   Lander, E. S., Green, P., Abrahamson, J., Barlow, A., Day, M. J.,    Lincoln, S. E. and Newberg, L. Genomics, 121, 185-199, 1987.-   Lazo, G. R., Pascal, A. S. and Ludwig, R. A., Bio/technology, 9:    963-967, 1991.-   Ledger, S. E., Delores, S. C. and Given, N. K. Plant Cell Reports,    10: 195-199, 1991.-   Liang, P. and Pardee, A. B. Science, 257: 967-971, 1992-   Liang, P., Averboukh, L. and Pardee, A. B. Nucl. Acids Res. 21:    3269-3275, 1993-   Marchuk, D., Drumm, M., Saulino, A., Collins, F. S. Nucl. Acids Res.    19: 1154, 1990-   Markham, K. R., Techniques of flavonoid identification. London:    Academic Press, 1982.-   Markham, K. R and Offman, D. J. Phytochem., 34: 679-685.-   Martin, C. and Gerats, T. In: The molecular biology of flowering.    (Jordan, B. R. ed), UK, CAB International, 219-255, 1993.-   McLean, M., Gerats, A. G. M., Baird, W. V. and Meagher, R. B. J.    Heredity 81: 341-346, 1990.-   Merrifield, J. Am. Chem. Soc. 85: 2149, 1964.-   Mizutani, M., Ward, E., DiMaio, J., Ohta, D., Ryals, J. and Sato, R.    Biochem. Biophys. Res. Comunn. 190: 875-880, 1993.-   Murashige, T. and Skoog, F., Physiol. Plant, 15: 73-97, 1962.-   Newman, T., de Bruijn, F. J., Green, P., Keegstra, K., Kende, H.,    McIntosh, L., Ohlrogge, J., Raikhel, N., Somerville, S.,    Thomashow, M. Plant Physiol. 106: 1241-1255, 1994.-   Pearson, W. R. and Lipman, D. J., Proc. Natl. Acad. Sci. USA 85:    2444-2448, 1988.-   Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A    Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory    Press, USA, 1989.-   Schenk, R. U. and Hilderbrandt, A. C., Can. J. Bot. 50: 199-204,    1972.-   Schram, A. W., Jonsson, L. M. V. and Bennink, G. J. H., Biochemistry    of flavonoid synthesis in Petunia hybrida. In: Petunia Sink, K. C.    (ed.), Springer-Verlag, Berlin, Germany, pp 68-75, 1984.-   Stafford, H. A., Flavonoid Metabolism. CRC Press, Inc. Boca Raton,    Fla., USA, 1990.-   Stotz, G. and Forkmann, G. Z. Naturforsch 37c: 19-23, 1982.-   Tabak, A. J. H., Meyer, H. and Bennink, G. J. H., Planta 139, 67-71,    1978.-   Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H. and    Matsubara, H. J. Biochem. 103: 954-961, 1988.-   Tanaka, Y., Yonekura, K., Fukuchi-Mizutani, M., Fukui, Y., Fujiwara,    H., Ashikari, T. and Kusumi, T. Plant Cell Physiol. 37(5): 711-716,    1996.-   Turpen, T. H. and Griffith, O. M. BioTechniques, 4: 11-15, 1986.-   van Tunen A. J. and Mol J. N. M. In: Plant Biotechnology    (Grierson, D. ed.) Glasgow: Blackie, 2: 9-31, 1990.-   Wiering, H. and de Vlaming, P., Inheritance and Biochemistry of    Pigments. In: Petunia Sink, K. C. (ed.), Springer-Verlag, Berlin,    Germany, pp 49-65, 1984.-   Wallroth, M., Gerats, A. G. M., Rogers, S. G., Fraley, R. T. and    Horsch, R. B., Mol. Gen. Genet. 202: 6-15, 1986.

1-39. (canceled)
 40. A DNA construct capable of reducing expression ofan endogenous gene encoding a flavonoid 3′-hydroxylase in a plant, saidDNA construct comprising a nucleotide sequence selected from the groupconsisting of: (1) a nucleotide sequence encoding an amino acid sequenceselected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
 21. SEQ ID NO: 23and SEQ ID NO: 25; and (2) a nucleotide sequence selected from, thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 20, SEQ ID NO: 22 and SEQID NO:
 24. 41. A transgenic plant having tissue exhibiting alteredcolour, said transgenic plant comprising a nucleic acid moleculeselected fro, the group consisting of: (1) a nucleotide sequenceencoding an amino acid sequence selected from the group consisting of:SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:
 21. SEQ ID NO: 23 and SEQ ID NO: 25; and (2) a nucleotidesequence selected fro, the group consisting of SEQ ID NO: 7, SEQ ID NO:20, SEQ ID NO: 22 and SEQ ID NO:
 24. 42. A cut flower from thetransgenic plant according to claim
 41. 43. A seed from the transgenicplant according to claim
 41. 44. A fruit from the transgenic plantaccording to claim
 41. 45. A leaf from the transgenic plant according toclaim 41.